Hmpv mrna vaccine composition

ABSTRACT

Provided herein are vaccine composition comprising a chemically-modified messenger ribonucleic acid (mRNA) encoding a hMPV fusion (F) glycoprotein and a chemically-modified mRNA encoding a hPIV3 F glycoprotein formulated in a cationic lipid nanoparticle formulation, and related method for inducing an antigen-specific immune response.

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) of U.S.provisional application 62/804,482, filed Feb. 12, 2019, U.S.provisional application 62/811,381, filed Feb. 27, 2019, and U.S.provisional application 62/877,937, filed Jul. 24, 2019, each of whichis herein incorporated by reference in its entirety.

BACKGROUND

Respiratory disease is a medical term that encompasses pathologicalconditions affecting the organs and tissues that make gas exchangepossible in higher organisms, and includes conditions of the upperrespiratory tract, trachea, bronchi, bronchioles, alveoli, pleura andpleural cavity, and the nerves and muscles of breathing. Respiratorydiseases range from mild and self-limiting, such as the common cold, tolife-threatening entities like bacterial pneumonia, pulmonary embolism,acute asthma and lung cancer. Respiratory disease is a common andsignificant cause of illness and death around the world. In the US,approximately 1 billion “common colds” occur each year. Respiratoryconditions are among the most frequent reasons for hospital stays amongchildren.

Despite decades of research, no vaccines currently exist (Sato andWright, Pediatr. Infect. Dis. J. 2008; 27 (10 Suppl):S123-5) forrespiratory virus, such as human metapneumovirus (hMPV) and humanparainfluenza virus type 3 (hPIV3). The continuing health problemsassociated with hMPV and hPIV3 are of concern internationally,reinforcing the importance of developing effective and safe vaccinecandidates against these viruses.

SUMMARY

Provided herein is a messenger ribonucleic acid (mRNA)-basedprophylactic vaccine comprising a mRNA encoding the full length hMPV Fglycoprotein and a mRNA encoding the full length hPIV3 F glycoprotein,which has been shown to be safe and effective for inducing aneutralizing antibody response specific for hMPV F glycoprotein andhPIV3 F glycoprotein. The vaccine should prevent upper and lowerrespiratory illnesses associated with hMPV and/or hPIV3 infection,particularly among young children and older adults. A principalimmunological goal is to boost functional antibody responses (serumneutralizing antibodies) along with cellular immune responses againstthese respiratory viruses. The mRNA vaccines provided herein include, insome embodiments, chemically modified mRNAs formulated within ionizablecationic lipid (e.g., Compound I)-containing lipid nanoparticles (LNPs).The mRNA vaccine is, in some embodiments, intramuscularly administeredin single dose annually prior to, or during, the cold season. To date,no effective vaccine to prevent hMPV or hPIV3 has been licensed, andtreatment is limited to supportive therapy.

Thus, some aspects of the present disclosure provide a method forproducing an antigen-specific immune response to human metapneumovirus(hMPV) and human parainfluenza virus (hPIV3) in a subject comprisingadministering to a human subject a safe and effective dose of a vaccinecomposition comprising a chemically-modified messenger ribonucleic acid(mRNA) encoding a hMPV fusion (F) glycoprotein and a chemically-modifiedmRNA encoding a hPIV3 F glycoprotein formulated in a lipid nanoparticlecomprising an ionizable cationic lipid, cholesterol, DSPC(1,2-Distearoyl-sn-glycero-3-phosphocholine), and optionally DMG-PEG(1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000), therebyinducing an antigen-specific immune response to hMPV and hPIV3 in thesubject.

In some aspects, the methods comprise administering to a human subject a25 μg to 100 μg dose of a vaccine composition comprising (a) achemically-modified messenger ribonucleic acid (mRNA) that encodes ahMPV fusion (F) glycoprotein and comprises an open reading frame thatcomprises a nucleotide sequence having at least 95% identity to thenucleotide sequence of SEQ ID NO: 7, and (b) a chemically-modified mRNAthat encodes a hPIV3 F glycoprotein and comprises an open reading framethat comprises a nucleotide sequence having at least 95% identity to thenucleotide sequence of SEQ ID NO: 9, formulated in a lipid nanoparticlecomprising 45-55 mole percent ionizable cationic lipid, 5-15 molepercent DSPC, 35-40 mole percent cholesterol, and optionally 1-2 molepercent DMG-PEG, thereby inducing an antigen-specific immune response tohMPV and hPIV3 in the subject.

In other aspects, the methods comprise administering to a human subjecta 25 μg to 100 μg dose of a vaccine composition comprising (a) achemically-modified messenger ribonucleic acid (mRNA) that encodes ahMPV fusion (F) glycoprotein and comprises an open reading frame thatcomprises the nucleotide sequence of SEQ ID NO: 7, and (b) achemically-modified mRNA that encodes a hPIV3 F glycoprotein andcomprises an open reading frame that comprises the nucleotide sequenceof SEQ ID NO: 9, formulated in formulated in an ionizable cationic lipidnanoparticle, thereby inducing an antigen-specific immune response tohMPV and hPIV3 in the subject, wherein the antigen-specific immuneresponse is measured as a geometric mean ratio (GMR) of serumneutralizing antibody titers to hMPV and hPIV3, the GMR for hMPV is inthe range of 6 to 6.5, and the GMR for hPIV3 is in the range of 3 to3.5.

In some embodiments, the open reading frame of (a) comprises thenucleotide sequence of SEQ ID NO: 7. In some embodiments, the mRNA of(a) comprises the nucleotide sequence of SEQ ID NO: 1. In someembodiments, the open reading frame of (b) comprises the nucleotidesequence of SEQ ID NO: 9. In some embodiments, the mRNA of (b) comprisesthe nucleotide sequence of SEQ ID NO: 2.

In some embodiments, the composition is administered at a dose of 25 μgto 300 μg, at a dose of 25 μg to 150 μg, at a dose of 25 μg to 100 μg,at a dose of 25 μg to 75 μg, or at a dose of 25 μg to 50 μg. In someembodiments, the composition is administered at a dose of 25 μg. In someembodiments, the composition is administered at a dose of 30 μg. In someembodiments, the composition is administered at a dose of 75 μg. In someembodiments, the composition is administered at a dose of 150 μg.

In some embodiments, the subject is an adult subject.

In some embodiments, the subject is a pediatric subject and thecomposition is administered at a dose of 10 μg to 150 μg, at a dose of10 μg to 100 μg, at a dose of 10 μg to 50 μg, or at a dose of 10 μg to30 μg. In some embodiments, the composition is administered at a dose of10 μg. In some embodiments, the composition is administered at a dose of30 μg. In some embodiments, the composition is administered at a dose of100 μg.

In some embodiments, administration of the vaccine composition elicitsserum neutralizing antibody titers against hMPV, including hMPV-A andhMPV-B, and hPIV3.

In some embodiments, administration of a single 10 μg, 25 μg, 30 μg, 75μg, 100 μg, 150 μg, or 300 μg dose of the vaccine composition elicitsserum neutralizing antibody titers against hMPV and hPIV3 with noapparent dose response.

In some embodiments, the antigen-specific immune response is measured asa geometric mean ratio (GMR) of serum neutralizing antibody titers tohMPV and hPIV3, and the GMR of 28 days to baseline titers for hMPV insubjects administered a ≥75 μg dose of the vaccine composition is in therange of 4 to 8, optionally 4.87-7.73. In some embodiments, the GMR forhMPV-A is 6.04. In some embodiments, the GMR for hMPV-B is 6.33.

In some embodiments, the antigen-specific immune response is measured asa geometric mean ratio (GMR) of serum neutralizing antibody titers tohMPV and hPIV3, and the GMR of 28 days to baseline titers for hPIV3 insubjects administered a ≥75 μg dose of the vaccine composition is in therange of 3 to 4, optionally 3.13-3.36. In some embodiments, the GMR forhPIV3 is 3.24.

In some embodiments, the antigen-specific immune response is measured asa geometric mean titer (GMT) of serum neutralizing antibodies to hMPV,and wherein the GMT in serum neutralizing antibodies to hMPV increasesin the subject at least 2 fold within 30 days relative to baseline. Insome embodiments, the antigen-specific immune response is measured as ageometric mean titer (GMT) of serum neutralizing antibodies to hMPV, andwherein the GMT in serum neutralizing antibodies to hMPV increases inthe subject at least 2 fold within 30 days relative to baseline,following a single 25 μg dose or a single 75 μg dose of the vaccinecomposition. In some embodiments, the antigen-specific immune responseis measured as a geometric mean titer (GMT) of serum neutralizingantibodies to hMPV, and wherein the GMT in serum neutralizing antibodiesto hMPV increases in the subject at least 6 fold within 30 days relativeto baseline. In some embodiments, the antigen-specific immune responseis measured as a GMT of serum neutralizing antibodies to hMPV, andwherein the GMT in serum neutralizing antibodies to hMPV increases inthe subject at least 6 fold within 30 days relative to baseline,following a single 25 μg dose or a single 75 μg dose of the vaccinecomposition.

In some embodiments, the antigen-specific immune response is measured asa GMT of serum neutralizing antibodies to hPIV3, and wherein the GMT inserum neutralizing antibodies to hPIV3 increases in the subject at least2 fold within 30 days relative to baseline. In some embodiments, theantigen-specific immune response is measured as a GMT of serumneutralizing antibodies to hPIV3, and wherein the GMT in serumneutralizing antibodies to hPIV3 increases in the subject at least 2fold within 30 days relative to baseline, following a single 25 μg doseor a single 75 μg dose of the vaccine composition. In some embodiments,the antigen-specific immune response is measured as a GMT of serumneutralizing antibodies to hPIV3, and wherein the GMT in serumneutralizing antibodies to hPIV3 increases in the subject at least 3fold within 30 days relative to baseline. In some embodiments, theantigen-specific immune response is measured as a GMT of serumneutralizing antibodies to hPIV3, and wherein the GMT in serumneutralizing antibodies to hPIV3 increases in the subject at least 3fold within 30 days relative to baseline, following a single 25 μg doseor a single 75 μg dose of the vaccine composition.

In some embodiments, administration of the vaccine composition elicitsserum neutralizing antibody titers against hMPV, including hMPV-A andhMPV-B, and hPIV3 that persist for at least 196 days postadministration.

In some embodiments, administration of the vaccine composition elicitsserum neutralizing antibody titers against hMPV, including hMPV-A andhMPV-B, that persist for at least 13 months post administration.

In some embodiments, administration of a second dose of the vaccinecomposition has negligible impact on the magnitude of hMPV or hPIV3serum neutralizing antibody titers.

In some embodiments, the ionizable cationic lipid comprises Compound I:

In some embodiments, the lipid nanoparticle comprises 45-55 mole percentionizable cationic lipid, 5-15 mole percent DSPC, 35-40 mole percentcholesterol, and optionally 1-2 mole percent DMG-PEG. In someembodiments, the lipid nanoparticle comprises 50 mole percent ionizablecationic lipid, 10 mole percent DSPC, 38.5 mole percent cholesterol, and1.5 mole percent DMG-PEG.

In some embodiments, the ratio of the mRNA encoding hMPV F glycoproteinto the mRNA encoding hPIV3 F glycoprotein in the vaccine composition is1:1.

In some embodiments, the mRNA encoding hMPV F glycoprotein and the mRNAencoding hPIV3 F glycoprotein comprise a 1-methylpseudourine chemicalmodification.

In some embodiments, the mRNA encoding hMPV F glycoprotein comprises anopen reading frame that comprises a nucleotide sequence having at least90% identity to the sequence of SEQ ID NO: 7. In some embodiments, themRNA encoding hMPV F glycoprotein comprises an open reading frame thatcomprises the nucleotide sequence of sequence of SEQ ID NO: 7. In someembodiments, the mRNA encoding hMPV F glycoprotein comprises anucleotide sequence having at least 90% identity to the sequence of SEQID NO: 1. In some embodiments, the mRNA encoding hMPV F glycoproteincomprises the nucleotide sequence of SEQ ID NO: 1.

In some embodiments, the mRNA encoding hPIV3 F glycoprotein comprises anopen reading frame that comprises a nucleotide sequence having at least90% identity to the sequence of SEQ ID NO: 9. In some embodiments, themRNA encoding hPIV3 F glycoprotein comprises an open reading frame thatcomprises the nucleotide sequence of sequence of SEQ ID NO: 9. In someembodiments, the mRNA encoding hPIV3 F glycoprotein comprises anucleotide sequence having at least 90% identity to the sequence of SEQID NO: 2. In some embodiments, the mRNA encoding hPIV3 F glycoproteincomprises the nucleotide sequence of SEQ ID NO: 2.

In some embodiments, the vaccine composition is administered viaintramuscular injection.

In some embodiments, the vaccine composition further comprises a mRNAencoding a respiratory syncytial virus (RSV) antigen formulated in alipid nanoparticle.

It should be understood that the vaccine compositions of the presentdisclosure are not naturally-occurring. That is, the RNA polynucleotidesencoding the respiratory virus antigens, as provided herein, do notoccur in nature. It should also be understood that the RNApolynucleotides described herein are isolated from viral proteins andviral lipids as they exist in nature. Thus, as provided herein, vaccinecomposition comprising an RNA formulated in a lipid nanoparticle, forexample, excludes viruses (i.e., the compositions are not, nor do theycontain, viruses).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts an overview of the study design. IST—internal safetyteam; SMC—safety monitoring committee

FIG. 2 depicts the dose-escalation phase of the study. BS—blood sample;‘visit’ denotes clinical visit.

FIG. 3 depicts the dose-selection phase of the study. BS—blood sample;‘visit’ denotes clinical visit.

FIGS. 4A-4C show graphs of neutralizing antibody (FIG. 4A, hMPV-A; FIG.4B, hMPV-B; FIG. 4C, PIV 3) by dose level and visit day; Pre-Protocol(PP) immunogenicity set.

FIGS. 5A-5C show graphs of neutralizing antibody (FIG. 5A, hMPV-A; FIG.5B, hMPV-B; FIG. 5C, PIV 3) by dose level (25 μg, 75 μg, 150 μg, or 300μg), regimen (1-dose vs. 2-dose) and visit day (day 1, day 28, day 56,and day 196 post first dose); PP immunogenicity set.

FIG. 6 shows the relationship between day 1 titer and response to firsthMPV/hPIV3 mRNA vaccination (day28/day 1 titer ratio); PP immunogenicityset.

FIGS. 7A-7C show neutralizing antibody kinetics by dose level andregimen (1-dose vs. 2-dose); PP immunogenicity set. FIG. 7A: hMPV-Aneutralizing antibody. FIG. 7B: hMPV-B neutralizing antibody. FIG. 7C:PIV3 neutralizing antibody.

FIG. 8 shows a schematic of the study design of a Phase 1b, randomized,observer-blinded, placebo-controlled, dose-ranging trial.

DETAILED DESCRIPTION

Results from the clinical trial data provided herein demonstrate that asingle hMPV/hPIV3 mRNA vaccination of the present disclosure elicited aboost in serum neutralization titers against both human metapneumovirus(hMPV) and human parainfluenza virus type 3 (hPIV3) at all dose levelstested (25, 75, 150 and 300 μg) with no apparent dose response. Thegeometric mean ratio (GMR) of Month 1 (28 days) to baseline titers indose groups>75 μg ranged from 4.87-7.73 for hMPV (e.g., 6.04 for hMPV-Aand 6.33 for hMPV-B) and from 3.13-3.36 for PIV3 (e.g., 3.24). A secondhMPV/hPIV3 mRNA vaccination at Month 1 had negligible impact on themagnitude of hMPV or PIV3 neutralization titers. PIV3 neutralizingantibody titers remained above baseline at all dose levels testedthrough Month 7 (the pooled GMR was 2.03 at Month 7), while hMPVneutralizing antibody titers remained above baseline at all dose levelstested through Month 13 (the pooled GMR was 1.87 for hMPV-A and 2.91 forhMPV-B). These results demonstrate that the hMPV/hPIV3 mRNA vaccineprovided herein is immunogenic at the lowest dose level tested and thatthe neutralizing antibody response reached a plateau. This hMPV/hPIV3mRNA vaccine is the first known vaccine targeting both hMPV and PIV3.The few other investigational vaccines that have been evaluatedtargeting either hMPV or PIV3 are all live-attenuated or chimericviruses, and none have induced significant antibody responses in adults.Given the similarities between hMPV, PIV3, and respiratory syncytialvirus (RSV), it is also informative to benchmark the hMPV/hPIV3 mRNAvaccination of the present disclosure against the many investigationalRSV vaccines that have been evaluated in adults. Such an analysissuggests that the hMPV/hPIV3 mRNA vaccine provided herein is at least asimmunogenic as most RSV vaccines (including RSV mRNA vaccines), with thePIV3 response roughly equivalent, and the hMPV response superior.

Only one hMPV vaccine has been evaluated in the clinic; the NationalInstitute of Allergy and Infectious Diseases (NIAID) evaluated alive-attenuated hMPV vaccine in a phase 1 study. A single dose wasadministered intranasally to adults, seropositive children andseronegative children. As expected, vaccine replication was restrictedin adults and seropositive children, and therefore did not induce anantibody response. However, the vaccine was also over-attenuated inseronegative children, and induced seroconversion in a minority of thesesubjects. It is thought that this program is still active, however thereare no known ongoing clinical studies.

Four different live-attenuated PIV3 vaccines have been developed byNIAID and tested in clinical studies. Two of these vaccines werepartnered with MedImmune (MEDI-560, MEDI-534), one of which alsoincludes the F antigen from RSV. All vaccines were administeredintranasally. Many (>10) phase 1 studies have been conducted over thepast ˜30 years, in populations including adults, seropositive children,seronegative children, and unscreened infants. A few progressed to phase2. Generalizing the results of these phase 1 and phase 2 studies: (1)vaccine replication was very restricted in adults and seropositivechildren and therefore little/no antibody response was induced; and (2)the vaccines induced seroconversion in most seronegative children, withbooster vaccine doses primarily increasing titers only in subjects witha low response to the first vaccination. These programs are not thoughtto be active, and there are no known ongoing clinical studies.

RSV is also responsible for substantial respiratory disease andassociated hospitalizations in infants and young children, with anincidence approximately the sum of hMPV +PIV3. RSV is closely related tohMPV and PIV3, and is a member of the Pneumoviradae family with hMPV.RSV also has a F protein on the viral surface that is highly conservedand is a major target of protective RSV neutralizing antibodies. Giventhe similarities between hMPV, PIV3 and RSV, it is worthwhile toconsider the immunogenicity of the hMPV/hPIV3 mRNA vaccine of thepresent disclosure relative to RSV vaccines. There is no licensedvaccine for RSV, but many institutions have investigational vaccines intheir pipelines, spanning phase 1 to phase 3 studies. A variety ofdifferent vaccine modalities are being evaluated, including subunitvaccines, nucleic acid-based vaccines (including an RSV mRNA vaccine),nanoparticles vaccines, vectored vaccines, and live-attenuated vaccines.All candidates were evaluated in adults that are all seropositive, andhumoral immunity is typically measured by RSV neutralizing antibody.Generally, most investigational RSV vaccines elicit a 2- to 5-fold risein baseline neutralizing antibody titers.

While it is difficult to directly compare virus neutralization titersacross institutions/studies/vaccines due to differences inneutralization assays and viruses. However, some general statements seemsupported by the data described herein. The hMPV/hPIV3 mRNA vaccine ofthe present disclosure is the first known vaccine targeting both hMPVand PIV3 to be tested in a clinical study; the neutralizing antibodyresponse elicited by the hMPV/hPIV3 mRNA vaccine in adults is superiorto that induced by the other investigational hMPV or PIV3 vaccinesevaluated in this age group; the boost in hMPV neutralization titerselicited by the hMPV/hPIV3 mRNA vaccine is superior to the boost in RSVneutralization titers by most RSV vaccines; and the boost in PIV3neutralization titers elicited by the hMPV/hPIV3 mRNA vaccine of thepresent disclosure is roughly equivalent.

Further, the Phase 1 safety findings of the hMPV/hPIV3 mRNA vaccineprovided herein in adults were generally consistent with Phase 1 safetyprofiles of other mRNA vaccines. These findings suggest that a doselevel of less than 300 μg has acceptable safety and tolerability.

Data from animal models and humans suggest that neutralizing antibodiesare important for protection against hMPV and PIV3, although correlateshave not been established in vaccine efficacy studies. The fusion (F)protein of hMPV and PIV3 are present on the viral surfaces, are highlyconserved on an amino acid level within each virus, and are dominanttargets of protective neutralizing antibodies. The hMPV/hPIV3 mRNAvaccine of the present disclosure, in some embodiments, includes twodistinct mRNA sequences that encode the full-length membrane-bound Fproteins of hMPV and PIV3, in a 1:1 target mass ratio. The mRNA vaccineprovided herein, which comprises mRNA encoding hMPV and mRNA encodinghPIV3 (e.g., on the same mRNA molecule or on separate mRNA molecules),is referred to as the “mRNA hMPV/hPIV3 vaccine.”

Infants acquire hMPV- and PIV3-specific antibodies from their mothers,and these antibodies are thought to provide substantial protectionagainst hMPV- and PIV3-associated respiratory disease during the veryfirst months of life. As maternal antibody wanes, diseaseincidence/severity tends to increase, followed by acquisition of naturalimmunity and a corresponding reduction of incidence/severity. Priorinfection does not always prevent re-infection, although diseaseseverity is typically less, particularly in immune competent healthyadults and older children. Secondary infections can boost neutralizingantibody titers, particularly if baseline titers are low. The vastmajority of adults have been infected at least once with both hMPV andPIV3, and therefore are seropositive as measured by serum neutralizingantibody.

As shown in the Examples herein, humoral immunity was assessed in thehMPV/hPIV3 mRNA vaccine by three different microneutralization assays,hMPV-A, hMPV-B, and PIV3. A and B are the two lineages of hMPV, and wereboth evaluated to investigate the breadth of antibodies elicited by thehMPV/hPIV3 mRNA vaccine. The F protein is well conserved between hMPV-Aand hMPV-B (˜95% amino acid identity), so it was thought that antibodiesprimed by one would neutralize both. Indeed, the hMPV/hPIV3 mRNA vaccineeffectively boosted antibodies that neutralized hMPV-A and hMPV-B.

Neutralizing antibodies against hMPV-A, hMPV-B, and PIV3 were detectedat baseline (Day 1, prior to vaccination) in all hMPV/hPIV3 mRNAvaccinated subjects. The magnitude of baseline neutralizing antibodytiters against hMPV-A and hMPV-B was similar (geometric mean titers[GMT]=3088.4 and 4453.2, respectively), given the ability of serumantibodies to cross-neutralize both hMPV lineages. The baselineneutralizing titer against PIV3 (GMT=378.4) was lower than that againsthMPV. However, comparison of titers between the hMPV and PIV3microneutralization assays was caveated by technical and biologicaldifferences in the assays and viruses. For example, hMPV forms foci ofinfected cells in culture, whereas PIV3 rapidly spreads throughout acell monolayer.

Human metapneumovirus (hMPV) is a negative-sense, single-stranded RNAvirus of the genus Pneumovirinae and of the family Paramyxoviridae andis closely related to the avian metapneumovirus (AMPV) subgroup C. Itwas isolated for the first time in 2001 in the Netherlands by using theRAP-PCR (RNA arbitrarily primed PCR) technique for identification ofunknown viruses growing in cultured cells. hMPV is second only to RSV asan important cause of viral lower respiratory tract illness (LRI) inyoung children. The seasonal epidemiology of hMPV appears to be similarto that of RSV, but the incidence of infection and illness appears to besubstantially lower. hMPV shares substantial homology with respiratorysyncytial virus in its surface glycoproteins. hMPV fusion (F)glycoprotein is related to other paramyxovirus fusion glycoproteins andappears to have homologous regions that may have similar functions. ThehMPV fusion glycoprotein amino acid sequence contains featurescharacteristic of other paramyxovirus F glycoproteins, including aputative cleavage site and potential N-linked glycosylation sites.Paramyxovirus fusion proteins are synthesized as inactive precursors(FO) that are cleaved by host cell proteases into the biologicallyfusion-active F1 and F2 domains (see, e.g., Cseke G. et al. Journal ofVirology 2007; 81 (2):698-707, incorporated herein by reference). Fusionglycoproteins are major antigenic determinants for all knownparamyxoviruses and for other viruses that possess similar fusionproteins such as human immunodeficiency virus, influenza virus, andEbola virus.

Human parainfluenza virus type 3 (hPIV3), like hMPV, is also anegative-sense, single-stranded sense RNA virus of the genusPneumovirinae and of the family Paramyxoviridae and is a major cause ofubiquitous acute respiratory infections of infancy and early childhood.Its incidence peaks around 4-12 months of age, and the virus isresponsible for 3-10% of hospitalizations, mainly for bronchiolitis andpneumonia. hPIV3 can be fatal, and in some instances is associated withneurologic diseases, such as febrile seizures. It can also result inairway remodeling, a significant cause of morbidity. In developingregions of the world, infants and young children are at the highest riskof mortality, either from primary hPIV3 viral infection or fromsecondary consequences, such as bacterial infections. hPIV3 Fglycoprotein is located on the viral envelope, where it facilitates theviral fusion and cell entry. The F glycoprotein is initially inactive,but proteolytic cleavage leads to its active forms, F1 and F2, which arelinked by disulfide bonds. This occurs when the HN protein binds itsreceptor on the host cell's surface. During early phases of infection,the F glycoprotein mediates penetration of the host cell by fusion ofthe viral envelope to the plasma membrane. In later stages of theinfection, the F glycoprotein facilitates the fusion of the infectedcells with neighboring uninfected cells, which leads to the formation ofa syncytium and spread of the infection.

It should be understood that the term “hMPV/hPIV3” encompasses “hMPV andhPIV3” as well as “hMPV or PIV3.”

Antigens

Antigens are proteins capable of inducing an immune response (e.g.,causing an immune system to produce antibodies against the antigens).Herein, use of the term antigen encompasses immunogenic proteins andimmunogenic fragments (an immunogenic fragment that induces (or iscapable of inducing) an immune response to hMPV/hPIV3), unless otherwisestated. It should be understood that the term “protein' encompassespeptides and the term “antigen” encompasses antigenic fragments.

The hMPV/hPIV3 antigens of the mRNA vaccine of the present disclosureare provided in the Sequence Listing elsewhere herein. In someembodiments, the hMPV/hPIV3 mRNA vaccine comprises a mRNA comprising theopen reading frame (ORF) sequence of SEQ ID NO: 7. In some embodiments,the hMPV/hPIV3 mRNA vaccine comprises a mRNA comprising the ORF sequenceof SEQ ID NO: 9. In some embodiments, the mRNA encoding the hMPV Fglycoprotein comprises the sequence of SEQ ID NO: 1. In someembodiments, the mRNA encoding the hPIV3 F glycoprotein comprises thesequence of SEQ ID NO: 2. In some embodiments, the hMPV F glycoproteincomprises the sequence of SEQ ID NO: 8. In some embodiments, the hPIV3 Fglycoprotein comprises the sequence of SEQ ID NO: 10. In someembodiments, the aforementioned mRNAs may further comprise a 5′ cap(e.g., 7mG(5′)ppp(5′)NlmpNp), a polyA tail (e.g., ˜100 nucleotides), ora 5′ cap and a polyA tail.

It should be understood that the hMPV/hPIV3 mRNA vaccine of the presentdisclosure may comprise a signal sequence. It should also be understoodthat the hMPV/hPIV3 mRNA vaccine of the present disclosure may includeany 5′ untranslated region (UTR) and/or any 3′ UTR. Exemplary UTRsequences are provided in the Sequence Listing; however, other UTRsequences (e.g., of the prior art) may be used or exchanged for any ofthe UTR sequences described herein. UTR₅ may also be omitted from thevaccine constructs provided herein.

Nucleic Acids

The hMPV/hPIV3 mRNA vaccine of the present disclosure comprise at leastone (one or more) ribonucleic acid (RNA) having an open reading frameencoding at least one hMPV/hPIV3 antigen. In some embodiments, the RNAis a messenger RNA (mRNA) having an open reading frame encoding at leastone hMPV/hPIV3 antigen. In some embodiments, the RNA (e.g., mRNA)further comprises a (at least one) 5′ UTR, 3′ UTR, a polyA tail and/or a5′ cap.

Nucleic acids comprise a polymer of nucleotides (nucleotide monomers),also referred to as polynucleotides. Nucleic acids may be or mayinclude, for example, deoxyribonucleic acids (DNAs), ribonucleic acids(RNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs),peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNAhaving a β-D-ribo configuration, α-LNA having an α-L-ribo configuration(a diastereomer of LNA), 2′-amino-LNA having a 2′-aminofunctionalization, and 2′-amino-α-LNA having a 2′-aminofunctionalization), ethylene nucleic acids (ENA), cyclohexenyl nucleicacids (CeNA) and/or chimeras and/or combinations thereof.

Messenger RNA (mRNA) is any ribonucleic acid that encodes a (at leastone) protein (a naturally-occurring, non-naturally-occurring, ormodified polymer of amino acids) and can be translated to produce theencoded protein in vitro, in vivo, in situ or ex vivo. The skilledartisan will appreciate that, except where otherwise noted, nucleic acidsequences set forth in the instant application may recite “T”s in arepresentative DNA sequence but where the sequence represents RNA (e.g.,mRNA), the “T”s would be substituted for “U”s. Thus, any of the DNAsdisclosed and identified by a particular sequence identification numberherein also disclose the corresponding RNA (e.g., mRNA) sequencecomplementary to the DNA, where each “T” of the DNA sequence issubstituted with “U.”

An open reading frame (ORF) is a continuous stretch of DNA or RNAbeginning with a start codon (e.g., methionine (ATG or AUG)) and endingwith a stop codon (e.g., TAA, TAG or TGA, or UAA, UAG or UGA). An ORFtypically encodes a protein. It will be understood that the sequencesdisclosed herein may further comprise additional elements, e.g., 5′ and3′ UTRs, but that those elements, unlike the ORF, need not necessarilybe present in a vaccine of the present disclosure.

Variants

In some embodiments, the hMPV/hPIV3 mRNA vaccine of the presentdisclosure encodes an hMPV/hPIV3 antigen variant. Antigen or otherpolypeptide variants refers to molecules that differ in their amino acidsequence from a wild-type, native or reference sequence. Theantigen/polypeptide variants may possess substitutions, deletions,and/or insertions at certain positions within the amino acid sequence,as compared to a native or reference sequence. Ordinarily, variantspossess at least 50% identity to a wild-type, native or referencesequence. In some embodiments, variants share at least 80%, or at least90% identity with a wild-type, native or reference sequence.

Variant antigens/polypeptides encoded by nucleic acids of the disclosuremay contain amino acid changes that confer any of a number of desirableproperties, e.g., that enhance their immunogenicity, enhance theirexpression, and/or improve their stability or PK/PD properties in asubject. Variant antigens/polypeptides can be made using routinemutagenesis techniques and assayed as appropriate to determine whetherthey possess the desired property. Assays to determine expression levelsand immunogenicity are well known in the art and exemplary such assaysare set forth in the Examples section Similarly, PK/PD properties of aprotein variant can be measured using art recognized techniques, e.g.,by determining expression of antigens in a vaccinated subject over timeand/or by looking at the durability of the induced immune response. Thestability of protein(s) encoded by a variant nucleic acid may bemeasured by assaying thermal stability or stability upon ureadenaturation or may be measured using in silico prediction. Methods forsuch experiments and in silico determinations are known in the art.

In some embodiments, a hMPV/hPIV3 mRNA vaccine comprises an mRNA ORFhaving a nucleotide sequence identified by any one of the sequencesprovided herein (see e.g., Sequence Listing), or having a nucleotidesequence at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% identical to anucleotide sequence identified by any one of the sequence providedherein.

The term “identity” refers to a relationship between the sequences oftwo or more polypeptides (e.g. antigens) or polynucleotides (nucleicacids), as determined by comparing the sequences. Identity also refersto the degree of sequence relatedness between or among sequences asdetermined by the number of matches between strings of two or more aminoacid residues or nucleic acid residues. Identity measures the percent ofidentical matches between the smaller of two or more sequences with gapalignments (if any) addressed by a particular mathematical model orcomputer program (e.g., “algorithms”). Identity of related antigens ornucleic acids can be readily calculated by known methods. “Percent (%)identity” as it applies to polypeptide or polynucleotide sequences isdefined as the percentage of residues (amino acid residues or nucleicacid residues) in the candidate amino acid or nucleic acid sequence thatare identical with the residues in the amino acid sequence or nucleicacid sequence of a second sequence after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent identity.Methods and computer programs for the alignment are well known in theart. It is understood that identity depends on a calculation of percentidentity but may differ in value due to gaps and penalties introduced inthe calculation. Generally, variants of a particular polynucleotide orpolypeptide (e.g., antigen) have at least 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% butless than 100% sequence identity to that particular referencepolynucleotide or polypeptide as determined by sequence alignmentprograms and parameters described herein and known to those skilled inthe art. Such tools for alignment include those of the BLAST suite(Stephen F. Altschul, et al (1997), “Gapped BLAST and PSI-BLAST: a newgeneration of protein database search programs”, Nucleic Acids Res.25:3389-3402). Another popular local alignment technique is based on theSmith-Waterman algorithm (Smith, T. F. & Waterman, M. S. (1981)“Identification of common molecular subsequences.” J. Mol. Biol.147:195-197). A general global alignment technique based on dynamicprogramming is the Needleman-Wunsch algorithm (Needleman, S. B. &Wunsch, C. D. (1970) “A general method applicable to the search forsimilarities in the amino acid sequences of two proteins.” J. Mol. Biol.48:443-453). More recently a Fast Optimal Global Sequence AlignmentAlgorithm (FOGSAA) has been developed that purportedly produces globalalignment of nucleotide and protein sequences faster than other optimalglobal alignment methods, including the Needleman-Wunsch algorithm.

As such, polynucleotides encoding peptides or polypeptides containingsubstitutions, insertions and/or additions, deletions and covalentmodifications with respect to reference sequences, in particular thepolypeptide (e.g., antigen) sequences disclosed herein, are includedwithin the scope of this disclosure. For example, sequence tags or aminoacids, such as one or more lysines, can be added to peptide sequences(e.g., at the N-terminal or C-terminal ends). Sequence tags can be usedfor peptide detection, purification or localization. Lysines can be usedto increase peptide solubility or to allow for biotinylation.Alternatively, amino acid residues located at the carboxy and aminoterminal regions of the amino acid sequence of a peptide or protein mayoptionally be deleted providing for truncated sequences. Certain aminoacids (e.g., C-terminal or N-terminal residues) may alternatively bedeleted depending on the use of the sequence, as for example, expressionof the sequence as part of a larger sequence which is soluble, or linkedto a solid support. In some embodiments, sequences for (or encoding)signal sequences, termination sequences, transmembrane domains, linkers,multimerization domains (such as, e.g., foldon regions) and the like maybe substituted with alternative sequences that achieve the same or asimilar function. In some embodiments, cavities in the core of proteinscan be filled to improve stability, e.g., by introducing larger aminoacids. In other embodiments, buried hydrogen bond networks may bereplaced with hydrophobic resides to improve stability. In yet otherembodiments, glycosylation sites may be removed and replaced withappropriate residues. Such sequences are readily identifiable to one ofskill in the art. It should also be understood that some of thesequences provided herein contain sequence tags or terminal peptidesequences (e.g., at the N-terminal or C-terminal ends) that may bedeleted, for example, prior to use in the preparation of an RNA (e.g.,mRNA) vaccine.

As recognized by those skilled in the art, protein fragments, functionalprotein domains, and homologous proteins are also considered to bewithin the scope of hMPV/hPIV3antigens of interest. For example,provided herein is any protein fragment (meaning a polypeptide sequenceat least one amino acid residue shorter than a reference antigensequence but otherwise identical) of a reference protein, provided thatthe fragment is immunogenic and confers a protective immune response tothe hMPV/hPIV3 pathogen. In addition to variants that are identical tothe reference protein but are truncated, in some embodiments, an antigenincludes 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations, as shown in anyof the sequences provided or referenced herein. Antigens/antigenicpolypeptides can range in length from about 4, 6, or 8 amino acids tofull length proteins.

Stabilizing Elements

Naturally-occurring eukaryotic mRNA molecules can contain stabilizingelements, including, but not limited to untranslated regions (UTR) attheir 5′-end (5′ UTR) and/or at their 3′-end (3′ UTR), in addition toother structural features, such as a 5′-cap structure or a 3′-poly(A)tail. Both the 5′ UTR and the 3′ UTR are typically transcribed from thegenomic DNA and are elements of the premature mRNA. Characteristicstructural features of mature mRNA, such as the 5′-cap and the3′-poly(A) tail are usually added to the transcribed (premature) mRNAduring mRNA processing.

In some embodiments, the hMPV/hPIV3 mRNA vaccine includes at least oneRNA polynucleotide having an open reading frame encoding at least oneantigenic polypeptide having at least one modification, at least one 5′terminal cap, and is formulated within a lipid nanoparticle. 5′-cappingof polynucleotides may be completed concomitantly during the invitro-transcription reaction using the following chemical RNA capanalogs to generate the 5′-guanosine cap structure according tomanufacturer protocols: 3′-O-Me-m7G(5′)ppp(5′) G [the ARCA cap];G(5′)ppp(5′)A; G(5′)ppp(5′)G; m7G(5′)ppp(5′)A; m7G(5′)ppp(5′)G (NewEngland BioLabs, Ipswich, Mass.). 5′-capping of modified RNA may becompleted post-transcriptionally using a Vaccinia Virus Capping Enzymeto generate the “Cap 0” structure: m7G(5′)ppp(5′)G (New England BioLabs,Ipswich, Mass.). Cap 1 structure may be generated using both VacciniaVirus Capping Enzyme and a 2′-O methyl-transferase to generate:m7G(5′)ppp(5′)G-2′-O-methyl. Cap 2 structure may be generated from theCap 1 structure followed by the 2′ methylation of the 5′-antepenultimatenucleotide using a 2′-O methyl-transferase. Cap 3 structure may begenerated from the Cap 2 structure followed by the 2′-O-methylation ofthe 5′-preantepenultimate nucleotide using a 2′-O methyl-transferase.Enzymes may be derived from a recombinant source.

The 3′-poly(A) tail is typically a stretch of adenine nucleotides addedto the 3′-end of the transcribed mRNA. It can, in some instances,comprise up to about 400 adenine nucleotides. In some embodiments, thelength of the 3′-poly(A) tail may be an essential element with respectto the stability of the individual mRNA.

In some embodiments, the hMPV/hPIV3 mRNA vaccine includes one or morestabilizing elements. Stabilizing elements may include for instance ahistone stem-loop. A stem-loop binding protein (SLBP), a 32 kDa proteinhas been identified. It is associated with the histone stem-loop at the3′-end of the histone messages in both the nucleus and the cytoplasm.Its expression level is regulated by the cell cycle; it peaks during theS-phase, when histone mRNA levels are also elevated. The protein hasbeen shown to be essential for efficient 3′-end processing of histonepre-mRNA by the U7 snRNP. SLBP continues to be associated with thestem-loop after processing, and then stimulates the translation ofmature histone mRNAs into histone proteins in the cytoplasm. The RNAbinding domain of SLBP is conserved through metazoa and protozoa; itsbinding to the histone stem-loop depends on the structure of the loop.The minimum binding site includes at least three nucleotides 5′ and twonucleotides 3′ relative to the stem-loop.

In some embodiments, the hMPV/hPIV3 mRNA vaccine includes a codingregion, at least one histone stem-loop, and optionally, a poly(A)sequence or polyadenylation signal. The poly(A) sequence orpolyadenylation signal generally should enhance the expression level ofthe encoded protein. The encoded protein, in some embodiments, is not ahistone protein, a reporter protein (e.g. Luciferase, GFP, EGFP,β-Galactosidase, EGFP), or a marker or selection protein (e.g.alpha-Globin, Galactokinase and Xanthine:guanine phosphoribosyltransferase (GPT)).

In some embodiments, the combination of a poly(A) sequence orpolyadenylation signal and at least one histone stem-loop, even thoughboth represent alternative mechanisms in nature, acts synergistically toincrease the protein expression beyond the level observed with either ofthe individual elements. The synergistic effect of the combination ofpoly(A) and at least one histone stem-loop does not depend on the orderof the elements or the length of the poly(A) sequence.

In some embodiments, the hMPV/hPIV3 mRNA vaccine does not comprise ahistone downstream element (HDE). “Histone downstream element” (HDE)includes a purine-rich polynucleotide stretch of approximately 15 to 20nucleotides 3′ of naturally occurring stem-loops, representing thebinding site for the U7 snRNA, which is involved in processing ofhistone pre-mRNA into mature histone mRNA. In some embodiments, thenucleic acid does not include an intron.

The hMPV/hPIV3 mRNA vaccine may or may not contain an enhancer and/orpromoter sequence, which may be modified or unmodified or which may beactivated or inactivated. In some embodiments, the histone stem-loop isgenerally derived from histone genes, and includes an intramolecularbase pairing of two neighbored partially or entirely reversecomplementary sequences separated by a spacer, consisting of a shortsequence, which forms the loop of the structure. The unpaired loopregion is typically unable to base pair with either of the stem loopelements. It occurs more often in RNA, as is a key component of many RNAsecondary structures, but may be present in single-stranded DNA as well.Stability of the stem-loop structure generally depends on the length,number of mismatches or bulges, and base composition of the pairedregion. In some embodiments, wobble base pairing (non-Watson-Crick basepairing) may result. In some embodiments, the at least one histonestem-loop sequence comprises a length of 15 to 45 nucleotides.

In some embodiments, the hMPV/hPIV3 mRNA vaccine has one or more AU-richsequences removed. These sequences, sometimes referred to as AURES aredestabilizing sequences found in the 3′UTR. The AURES may be removedfrom the RNA vaccines. Alternatively the AURES may remain in the RNAvaccine.

Signal Peptides

In some embodiments, a hMPV/hPIV3 mRNA vaccine comprises a mRNA havingan ORF that encodes a signal peptide fused to the hMPV/hPIV3 antigen.Signal peptides, comprising the N-terminal 15-60 amino acids ofproteins, are typically needed for the translocation across the membraneon the secretory pathway and, thus, universally control the entry ofmost proteins both in eukaryotes and prokaryotes to the secretorypathway. In eukaryotes, the signal peptide of a nascent precursorprotein (pre-protein) directs the ribosome to the rough endoplasmicreticulum (ER) membrane and initiates the transport of the growingpeptide chain across it for processing. ER processing produces matureproteins, wherein the signal peptide is cleaved from precursor proteins,typically by a ER-resident signal peptidase of the host cell, or theyremain uncleaved and function as a membrane anchor. A signal peptide mayalso facilitate the targeting of the protein to the cell membrane.

A signal peptide may have a length of 15-60 amino acids. For example, asignal peptide may have a length of 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,or 60 amino acids. In some embodiments, a signal peptide has a length of20-60, 25-60, 30-60, 35-60, 40-60, 45-60, 50-60, 55-60, 15-55, 20-55,25-55, 30-55, 35-55, 40-55, 45-55, 50-55, 15-50, 20-50, 25-50, 30-50,35-50, 40-50, 45-50, 15-45, 20-45, 25-45, 30-45, 35-45, 40-45, 15-40,20-40, 25-40, 30-40, 35-40, 15-35, 20-35, 25-35, 30-35, 15-30, 20-30,25-30, 15-25, 20-25, or 15-20 amino acids.

Signal peptides from heterologous genes (which regulate expression ofgenes other than hMPV/hPIV3 antigens in nature) are known in the art andcan be tested for desired properties and then incorporated into anucleic acid of the disclosure. In some embodiments, the signal peptidemay comprise one of the following sequences:

(SEQ ID NO: 11) MDSKGSSQKGSRLLLLLVVSNLLLPQGVVG, (SEQ ID NO: 12)MDWTWILFLVAAATRVHS; (SEQ ID NO: 13) METPAQLLFLLLLWLPDTTG;(SEQ ID NO: 14) MLGSNSGQRVVFTILLLLVAPAYS; (SEQ ID NO: 15)MKCLLYLAFLFIGVNCA; (SEQ ID NO: 16) MWLVSLAIVTACAGA.

Fusion Proteins

In some embodiments, the hMPV/hPIV3 mRNA vaccine of the presentdisclosure includes a mRNA encoding an antigenic fusion protein. Thus,the encoded antigen or antigens may include two or more proteins (e.g.,protein and/or protein fragment) joined together. In some embodiments,the mRNA encodes a hMPV F glycoprotein fused to a hPIV3 F glycoprotein.Alternatively, the protein to which a protein antigen is fused does notpromote a strong immune response to itself, but rather to the hMPV/hPIV3antigen. Antigenic fusion proteins, in some embodiments, retain thefunctional property from each original protein.

Scaffold Moieties

The RNA (e.g., mRNA) vaccines as provided herein, in some embodiments,encode fusion proteins that comprise hMPV/hPIV3 antigens linked toscaffold moieties. In some embodiments, such scaffold moieties impartdesired properties to an antigen encoded by a nucleic acid of thedisclosure. For example scaffold proteins may improve the immunogenicityof an antigen, e.g., by altering the structure of the antigen, alteringthe uptake and processing of the antigen, and/or causing the antigen tobind to a binding partner.

In some embodiments, the scaffold moiety is protein that canself-assemble into protein nanoparticles that are highly symmetric,stable, and structurally organized, with diameters of 10-150 nm, ahighly suitable size range for optimal interactions with various cellsof the immune system. In some embodiments, viral proteins or virus-likeparticles can be used to form stable nanoparticle structures. Examplesof such viral proteins are known in the art. For example, in someembodiments, the scaffold moiety is a hepatitis B surface antigen(HBsAg). HBsAg forms spherical particles with an average diameter of ˜22nm and which lacked nucleic acid and hence are non-infectious(Lopez-Sagaseta, J. et al. Computational and Structural BiotechnologyJournal 14 (2016) 58-68). In some embodiments, the scaffold moiety is ahepatitis B core antigen (HBcAg) self-assembles into particles of 24-31nm diameter, which resembled the viral cores obtained from HBV-infectedhuman liver. HBcAg produced in self-assembles into two classes ofdifferently sized nanoparticles of 300 Å and 360 Å diameter,corresponding to 180 or 240 protomers. In some embodiments thehMPV/hPIV3 antigen is fused to HBsAG or HBcAG to facilitateself-assembly of nanoparticles displaying the hMPV/hPIV3 antigen.

In another embodiment, bacterial protein platforms may be used.Non-limiting examples of these self-assembling proteins includeferritin, lumazine and encapsulin.

Ferritin is a protein whose main function is intracellular iron storage.Ferritin is made of 24 subunits, each composed of a four-alpha-helixbundle, that self-assemble in a quaternary structure with octahedralsymmetry (Cho K. J. et al. J Mol Biol. 2009; 390:83-98). Severalhigh-resolution structures of ferritin have been determined, confirmingthat Helicobacter pylori ferritin is made of 24 identical protomers,whereas in animals, there are ferritin light and heavy chains that canassemble alone or combine with different ratios into particles of 24subunits (Granier T. et al. J Biol Inorg Chem. 2003; 8:105-111; LawsonD. M. et al. Nature. 1991; 349:541-544). Ferritin self-assembles intonanoparticles with robust thermal and chemical stability. Thus, theferritin nanoparticle is well-suited to carry and expose antigens.

Lumazine synthase (LS) is also well-suited as a nanoparticle platformfor antigen display. LS, which is responsible for the penultimatecatalytic step in the biosynthesis of riboflavin, is an enzyme presentin a broad variety of organisms, including archaea, bacteria, fungi,plants, and eubacteria (Weber S. E. Flavins and Flavoproteins. Methodsand Protocols, Series: Methods in Molecular Biology. 2014). The LSmonomer is 150 amino acids long, and consists of beta-sheets along withtandem alpha-helices flanking its sides. A number of differentquaternary structures have been reported for LS, illustrating itsmorphological versatility: from homopentamers up to symmetricalassemblies of 12 pentamers forming capsids of 150 A diameter. Even LScages of more than 100 subunits have been described (Zhang X. et al. JMol Biol. 2006; 362:753-770).

Encapsulin, a novel protein cage nanoparticle isolated from thermophileThermotoga maritima, may also be used as a platform to present antigenson the surface of self-assembling nanoparticles. Encapsulin is assembledfrom 60 copies of identical 31 kDa monomers having a thin andicosahedral T=1 symmetric cage structure with interior and exteriordiameters of 20 and 24 nm, respectively (Sutter M. et al. Nat Struct MolBiol. 2008, 15: 939-947). Although the exact function of encapsulin inT. maritima is not clearly understood yet, its crystal structure hasbeen recently solved and its function was postulated as a cellularcompartment that encapsulates proteins such as DyP (Dye decolorizingperoxidase) and Flp (Ferritin like protein), which are involved inoxidative stress responses (Rahmanpour R. et al. FEBS J. 2013, 280:2097-2104).

Linkers and Cleavable Peptides

In some embodiments, the mRNAs of the disclosure encode more than onepolypeptide, referred to herein as fusion proteins. In some embodiments,the mRNA further encodes a linker located between at least one or eachdomain of the fusion protein. The linker can be, for example, acleavable linker or protease-sensitive linker. In some embodiments, thelinker is selected from the group consisting of F2A linker, P2A linker,T2A linker, E2A linker, and combinations thereof. This family ofself-cleaving peptide linkers, referred to as 2A peptides, has beendescribed in the art (see for example, Kim, J. H. et al. (2011) PLoS ONE6:e18556). In some embodiments, the linker is an F2A linker. In someembodiments, the linker is a GGGS linker. In some embodiments, thefusion protein contains three domains with intervening linkers, havingthe structure: domain-linker-domain-linker-domain.

Cleavable linkers known in the art may be used in connection with thedisclosure. Exemplary such linkers include: F2A linkers, T2A linkers,P2A linkers, E2A linkers (See, e.g., WO2017127750). The skilled artisanwill appreciate that other art-recognized linkers may be suitable foruse in the constructs of the disclosure (e.g., encoded by the nucleicacids of the disclosure). The skilled artisan will likewise appreciatethat other polycistronic constructs (mRNA encoding more than oneantigen/polypeptide separately within the same molecule) may be suitablefor use as provided herein.

Sequence Optimization

In some embodiments, an ORF encoding an antigen of the disclosure iscodon optimized. Codon optimization methods are known in the art. Forexample, an ORF of any one or more of the sequences provided herein maybe codon optimized. Codon optimization, in some embodiments, may be usedto match codon frequencies in target and host organisms to ensure properfolding; bias GC content to increase mRNA stability or reduce secondarystructures; minimize tandem repeat codons or base runs that may impairgene construction or expression; customize transcriptional andtranslational control regions; insert or remove protein traffickingsequences; remove/add post translation modification sites in encodedprotein (e.g., glycosylation sites); add, remove or shuffle proteindomains; insert or delete restriction sites; modify ribosome bindingsites and mRNA degradation sites; adjust translational rates to allowthe various domains of the protein to fold properly; or reduce oreliminate problem secondary structures within the polynucleotide. Codonoptimization tools, algorithms and services are known in theart—non-limiting examples include services from GeneArt (LifeTechnologies), DNA2.0 (Menlo Park Calif.) and/or proprietary methods. Insome embodiments, the open reading frame (ORF) sequence is optimizedusing optimization algorithms.

In some embodiments, a codon optimized sequence shares less than 95%sequence identity to a naturally-occurring or wild-type sequence ORF(e.g., a naturally-occurring or wild-type mRNA sequence encoding ahMPV/hPIV3 antigen). In some embodiments, a codon optimized sequenceshares less than 90% sequence identity to a naturally-occurring orwild-type sequence (e.g., a naturally-occurring or wild-type mRNAsequence encoding a hMPV/hPIV3 antigen). In some embodiments, a codonoptimized sequence shares less than 85% sequence identity to anaturally-occurring or wild-type sequence (e.g., a naturally-occurringor wild-type mRNA sequence encoding a hMPV/hPIV3 antigen). In someembodiments, a codon optimized sequence shares less than 80% sequenceidentity to a naturally-occurring or wild-type sequence (e.g., anaturally-occurring or wild-type mRNA sequence encoding a hMPV/hPIV3antigen). In some embodiments, a codon optimized sequence shares lessthan 75% sequence identity to a naturally-occurring or wild-typesequence (e.g., a naturally-occurring or wild-type mRNA sequenceencoding a hMPV/hPIV3 antigen).

In some embodiments, a codon optimized sequence shares between 65% and85% (e.g., between about 67% and about 85% or between about 67% andabout 80%) sequence identity to a naturally-occurring or wild-typesequence (e.g., a naturally-occurring or wild-type mRNA sequenceencoding a hMPV/hPIV3 antigen). In some embodiments, a codon optimizedsequence shares between 65% and 75% or about 80% sequence identity to anaturally-occurring or wild-type sequence (e.g., a naturally-occurringor wild-type mRNA sequence encoding a hMPV/hPIV3 antigen).

In some embodiments, a codon-optimized sequence encodes an antigen thatis as immunogenic as, or more immunogenic than (e.g., at least 10%, atleast 20%, at least 30%, at least 40%, at least 50%, at least 100%, orat least 200% more), than a hMPV/hPIV3 antigen encoded by anon-codon-optimized sequence.

When transfected into mammalian host cells, the modified mRNAs have astability of between 12-18 hours, or greater than 18 hours, e.g., 24,36, 48, 60, 72, or greater than 72 hours and are capable of beingexpressed by the mammalian host cells.

In some embodiments, a codon optimized RNA may be one in which thelevels of G/C are enhanced. The G/C-content of nucleic acid molecules(e.g., mRNA) may influence the stability of the RNA. RNA having anincreased amount of guanine (G) and/or cytosine (C) residues may befunctionally more stable than RNA containing a large amount of adenine(A) and thymine (T) or uracil (U) nucleotides. As an example,WO02/098443 discloses a pharmaceutical composition containing an mRNAstabilized by sequence modifications in the translated region. Due tothe degeneracy of the genetic code, the modifications work bysubstituting existing codons for those that promote greater RNAstability without changing the resulting amino acid. The approach islimited to coding regions of the RNA.

Chemically Unmodified Nucleotides

In some embodiments, at least one RNA (e.g., mRNA) of the hMPV/hPIV3mRNA vaccine of the present disclosure is not chemically modified andcomprises the standard ribonucleotides consisting of adenosine,guanosine, cytosine and uridine. In some embodiments, nucleotides andnucleosides of the present disclosure comprise standard nucleosideresidues such as those present in transcribed RNA (e.g. A, G, C, or U).In some embodiments, nucleotides and nucleosides of the presentdisclosure comprise standard deoxyribonucleosides such as those presentin DNA (e.g. dA, dG, dC, or dT).

Chemical Modifications

The hMPV/hPIV3 mRNA vaccine of the present disclosure comprise, in someembodiments, at least one nucleic acid (e.g., RNA) having an openreading frame encoding at least one hMPV/hPIV3 antigen, wherein thenucleic acid comprises nucleotides and/or nucleosides that can bestandard (unmodified) or modified as is known in the art. In someembodiments, nucleotides and nucleosides of the present disclosurecomprise modified nucleotides or nucleosides. Such modified nucleotidesand nucleosides can be naturally-occurring modified nucleotides andnucleosides or non-naturally occurring modified nucleotides andnucleosides. Such modifications can include those at the sugar,backbone, or nucleobase portion of the nucleotide and/or nucleoside asare recognized in the art.

In some embodiments, a naturally-occurring modified nucleotide ornucleotide of the disclosure is one as is generally known or recognizedin the art. Non-limiting examples of such naturally occurring modifiednucleotides and nucleotides can be found, inter alia, in the widelyrecognized MODOMICS database.

In some embodiments, a non-naturally occurring modified nucleotide ornucleoside of the disclosure is one as is generally known or recognizedin the art. Non-limiting examples of such non-naturally occurringmodified nucleotides and nucleosides can be found, inter alia, inpublished US application Nos. PCT/US2012/058519; PCT/US2013/075177;PCT/US2014/058897; PCT/U52014/058891; PCT/U52014/070413;PCT/US2015/36773; PCT/US2015/36759; PCT/US2015/36771; orPCT/IB2017/051367 all of which are incorporated by reference herein.

Hence, nucleic acids of the disclosure (e.g., DNA nucleic acids and RNAnucleic acids, such as mRNA nucleic acids) can comprise standardnucleotides and nucleosides, naturally-occurring nucleotides andnucleosides, non-naturally-occurring nucleotides and nucleosides, or anycombination thereof.

Nucleic acids of the disclosure (e.g., DNA nucleic acids and RNA nucleicacids, such as mRNA nucleic acids), in some embodiments, comprisevarious (more than one) different types of standard and/or modifiednucleotides and nucleosides. In some embodiments, a particular region ofa nucleic acid contains one, two or more (optionally different) types ofstandard and/or modified nucleotides and nucleosides.

In some embodiments, a modified RNA nucleic acid (e.g., a modified mRNAnucleic acid), introduced to a cell or organism, exhibits reduceddegradation in the cell or organism, respectively, relative to anunmodified nucleic acid comprising standard nucleotides and nucleosides.

In some embodiments, a modified RNA nucleic acid (e.g., a modified mRNAnucleic acid), introduced into a cell or organism, may exhibit reducedimmunogenicity in the cell or organism, respectively (e.g., a reducedinnate response) relative to an unmodified nucleic acid comprisingstandard nucleotides and nucleosides.

Nucleic acids (e.g., RNA nucleic acids, such as mRNA nucleic acids), insome embodiments, comprise non-natural modified nucleotides that areintroduced during synthesis or post-synthesis of the nucleic acids toachieve desired functions or properties. The modifications may bepresent on internucleotide linkages, purine or pyrimidine bases, orsugars. The modification may be introduced with chemical synthesis orwith a polymerase enzyme at the terminal of a chain or anywhere else inthe chain. Any of the regions of a nucleic acid may be chemicallymodified.

The present disclosure provides for modified nucleosides and nucleotidesof a nucleic acid (e.g., RNA nucleic acids, such as mRNA nucleic acids).A “nucleoside” refers to a compound containing a sugar molecule (e.g., apentose or ribose) or a derivative thereof in combination with anorganic base (e.g., a purine or pyrimidine) or a derivative thereof(also referred to herein as “nucleobase”). A “nucleotide” refers to anucleoside, including a phosphate group. Modified nucleotides may bysynthesized by any useful method, such as, for example, chemically,enzymatically, or recombinantly, to include one or more modified ornon-natural nucleosides. Nucleic acids can comprise a region or regionsof linked nucleosides. Such regions may have variable backbone linkages.The linkages can be standard phosphodiester linkages, in which case thenucleic acids would comprise regions of nucleotides.

Modified nucleotide base pairing encompasses not only the standardadenosine-thymine, adenosine-uracil, or guanosine-cytosine base pairs,but also base pairs formed between nucleotides and/or modifiednucleotides comprising non-standard or modified bases, wherein thearrangement of hydrogen bond donors and hydrogen bond acceptors permitshydrogen bonding between a non-standard base and a standard base orbetween two complementary non-standard base structures, such as, forexample, in those nucleic acids having at least one chemicalmodification. One example of such non-standard base pairing is the basepairing between the modified nucleotide inosine and adenine, cytosine oruracil. Any combination of base/sugar or linker may be incorporated intonucleic acids of the present disclosure.

In some embodiments, modified nucleobases in nucleic acids (e.g., RNAnucleic acids, such as mRNA nucleic acids) comprise1-methyl-pseudouridine (m1ψ), 1-ethyl-pseudouridine (e1ψ),5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C), and/or pseudouridine(ψ). In some embodiments, modified nucleobases in nucleic acids (e.g.,RNA nucleic acids, such as mRNA nucleic acids) comprise 5-methoxymethyluridine, 5-methylthio uridine, 1-methoxymethyl pseudouridine, 5-methylcytidine, and/or 5-methoxy cytidine. In some embodiments, thepolyribonucleotide includes a combination of at least two (e.g., 2, 3, 4or more) of any of the aforementioned modified nucleobases, includingbut not limited to chemical modifications.

In some embodiments, a mRNA of the disclosure comprises1-methyl-pseudouridine (m1ψ) substitutions at one or more or all uridinepositions of the nucleic acid.

In some embodiments, a mRNA of the disclosure comprises1-methyl-pseudouridine (m1ψ) substitutions at one or more or all uridinepositions of the nucleic acid and 5-methyl cytidine substitutions at oneor more or all cytidine positions of the nucleic acid.

In some embodiments, a mRNA of the disclosure comprises pseudouridine(w) substitutions at one or more or all uridine positions of the nucleicacid.

In some embodiments, a mRNA of the disclosure comprises pseudouridine(w) substitutions at one or more or all uridine positions of the nucleicacid and 5-methyl cytidine substitutions at one or more or all cytidinepositions of the nucleic acid.

In some embodiments, a mRNA of the disclosure comprises uridine at oneor more or all uridine positions of the nucleic acid.

In some embodiments, mRNAs are uniformly modified (e.g., fully modified,modified throughout the entire sequence) for a particular modification.For example, a nucleic acid can be uniformly modified with1-methyl-pseudouridine, meaning that all uridine residues in the mRNAsequence are replaced with 1-methyl-pseudouridine. Similarly, a nucleicacid can be uniformly modified for any type of nucleoside residuepresent in the sequence by replacement with a modified residue such asthose set forth above.

The nucleic acids of the present disclosure may be partially or fullymodified along the entire length of the molecule. For example, one ormore or all or a given type of nucleotide (e.g., purine or pyrimidine,or any one or more or all of A, G, U, C) may be uniformly modified in anucleic acid of the disclosure, or in a predetermined sequence regionthereof (e.g., in the mRNA including or excluding the polyA tail). Insome embodiments, all nucleotides X in a nucleic acid of the presentdisclosure (or in a sequence region thereof) are modified nucleotides,wherein X may be any one of nucleotides A, G, U, C, or any one of thecombinations A+G, A+U, A+C, G+U, G+C, U+C, A+G+U, A+G+C, G+U+C or A+G+C.

The nucleic acid may contain from about 1% to about 100% modifiednucleotides (either in relation to overall nucleotide content, or inrelation to one or more types of nucleotide, i.e., any one or more of A,G, U or C) or any intervening percentage (e.g., from 1% to 20%, from 1%to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%,from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10%to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%,from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%,from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%,from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to 100%,from 80% to 90%, from 80% to 95%, from 80% to 100%, from 90% to 95%,from 90% to 100%, and from 95% to 100%). It will be understood that anyremaining percentage is accounted for by the presence of unmodified A,G, U, or C.

The mRNAs may contain at a minimum 1% and at maximum 100% modifiednucleotides, or any intervening percentage, such as at least 5% modifiednucleotides, at least 10% modified nucleotides, at least 25% modifiednucleotides, at least 50% modified nucleotides, at least 80% modifiednucleotides, or at least 90% modified nucleotides. For example, thenucleic acids may contain a modified pyrimidine such as a modifieduracil or cytosine. In some embodiments, at least 5%, at least 10%, atleast 25%, at least 50%, at least 80%, at least 90% or 100% of theuracil in the nucleic acid is replaced with a modified uracil (e.g., a5-substituted uracil). The modified uracil can be replaced by a compoundhaving a single unique structure, or can be replaced by a plurality ofcompounds having different structures (e.g., 2, 3, 4 or more uniquestructures). In some embodiments, at least 5%, at least 10%, at least25%, at least 50%, at least 80%, at least 90% or 100% of the cytosine inthe nucleic acid is replaced with a modified cytosine (e.g., a5-substituted cytosine). The modified cytosine can be replaced by acompound having a single unique structure, or can be replaced by aplurality of compounds having different structures (e.g., 2, 3, 4 ormore unique structures).

Untranslated Regions (UTRs)

The mRNAs of the present disclosure may comprise one or more regions orparts which act or function as an untranslated region. Where mRNAs aredesigned to encode at least one antigen of interest, the nucleic maycomprise one or more of these untranslated regions (UTRs). Wild-typeuntranslated regions of a nucleic acid are transcribed but nottranslated. In mRNA, the 5′ UTR starts at the transcription start siteand continues to the start codon but does not include the start codon;whereas, the 3′ UTR starts immediately following the stop codon andcontinues until the transcriptional termination signal. There is growingbody of evidence about the regulatory roles played by the UTR₅ in termsof stability of the nucleic acid molecule and translation. Theregulatory features of a UTR can be incorporated into thepolynucleotides of the present disclosure to, among other things,enhance the stability of the molecule. The specific features can also beincorporated to ensure controlled down-regulation of the transcript incase they are misdirected to undesired organs sites. A variety of 5′UTRand 3′UTR sequences are known and available in the art.

A 5′ UTR is region of an mRNA that is directly upstream (5′) from thestart codon (the first codon of an mRNA transcript translated by aribosome). A 5′ UTR does not encode a protein (is non-coding). Natural5′UTR₅ have features that play roles in translation initiation. Theyharbor signatures like Kozak sequences which are commonly known to beinvolved in the process by which the ribosome initiates translation ofmany genes. Kozak sequences have the consensus CCR(A/G)CCAUGG (SEQ IDNO: 17), where R is a purine (adenine or guanine) three bases upstreamof the start codon (AUG), which is followed by another ‘G’.5′UTR alsohave been known to form secondary structures which are involved inelongation factor binding.

In some embodiments of the disclosure, a 5′ UTR is a heterologous UTR,i.e., is a UTR found in nature associated with a different ORF. Inanother embodiment, a 5′ UTR is a synthetic UTR, i.e., does not occur innature. Synthetic UTR₅ include UTR₅ that have been mutated to improvetheir properties, e.g., which increase gene expression as well as thosewhich are completely synthetic. Exemplary 5′ UTR₅ include Xenopus orhuman derived a-globin or b-globin (U.S. Pat. Nos. 8,278,063;9,012,219), human cytochrome b-245 a polypeptide, and hydroxysteroid(17b) dehydrogenase, and Tobacco etch virus (U.S. Pat. Nos. 8,278,063,9,012,219). CMV immediate-early 1 (IE1) gene (US20140206753,WO2013/185069), the sequence GGGAUCCUACC (SEQ ID NO: 18) (WO2014144196)may also be used. In another embodiment, 5′ UTR of a TOP gene is a 5′UTR of a TOP gene lacking the 5′ TOP motif (the oligopyrimidine tract)(e.g., WO/2015101414, WO2015101415, WO/2015/062738, WO2015024667,WO2015024667; 5′ UTR element derived from ribosomal protein Large 32(L32) gene (WO/2015101414, WO2015101415, WO/2015/062738), 5′ UTR elementderived from the 5′UTR of an hydroxysteroid (1743) dehydrogenase 4 gene(HSD17B4) (WO2015024667), or a 5′ UTR element derived from the 5′ UTR ofATP5A1 (WO2015024667) can be used. In some embodiments, an internalribosome entry site (IRES) is used instead of a 5′ UTR.

In some embodiments, a 5′ UTR of the present disclosure comprises asequence selected from SEQ ID NO: 3 and SEQ ID NO: 4.

A 3′ UTR is region of an mRNA that is directly downstream (3′) from thestop codon (the codon of an mRNA transcript that signals a terminationof translation). A 3′ UTR does not encode a protein (is non-coding).Natural or wild type 3′ UTR₅ are known to have stretches of adenosinesand uridines embedded in them. These AU rich signatures are particularlyprevalent in genes with high rates of turnover. Based on their sequencefeatures and functional properties, the AU rich elements (AREs) can beseparated into three classes (Chen et al, 1995): Class I AREs containseveral dispersed copies of an AUUUA motif within U-rich regions. C-Mycand MyoD contain class I AREs. Class II AREs possess two or moreoverlapping UUAUUUA(U/A)(U/A) (SEQ ID NO: 19) nonamers. Moleculescontaining this type of AREs include GM-CSF and TNF-a. Class III ARESare less well defined. These U rich regions do not contain an AUUUAmotif. c-Jun and Myogenin are two well-studied examples of this class.Most proteins binding to the AREs are known to destabilize themessenger, whereas members of the ELAV family, most notably HuR, havebeen documented to increase the stability of mRNA. HuR binds to AREs ofall the three classes. Engineering the HuR specific binding sites intothe 3′ UTR of nucleic acid molecules will lead to HuR binding and thus,stabilization of the message in vivo.

Introduction, removal or modification of 3′ UTR AU rich elements (AREs)can be used to modulate the stability of nucleic acids (e.g., RNA) ofthe disclosure. When engineering specific nucleic acids, one or morecopies of an ARE can be introduced to make nucleic acids of thedisclosure less stable and thereby curtail translation and decreaseproduction of the resultant protein. Likewise, AREs can be identifiedand removed or mutated to increase the intracellular stability and thusincrease translation and production of the resultant protein.Transfection experiments can be conducted in relevant cell lines, usingnucleic acids of the disclosure and protein production can be assayed atvarious time points post-transfection. For example, cells can betransfected with different ARE-engineering molecules and by using anELISA kit to the relevant protein and assaying protein produced at 6hour, 12 hour, 24 hour, 48 hour, and 7 days post-transfection.

3′ UTR₅ may be heterologous or synthetic. With respect to 3′ UTRs,globin UTRs, including Xenopus β-globin UTR₅ and human β-globin UTR₅ areknown in the art (U.S. Pat. Nos. 8,278,063, 9,012,219, US20110086907). Amodified β-globin construct with enhanced stability in some cell typesby cloning two sequential human β-globin 3′UTR₅ head to tail has beendeveloped and is well known in the art (US2012/0195936, WO2014/071963).In addition a2-globin, a1-globin, UTR₅ and mutants thereof are alsoknown in the art (WO2015101415, WO2015024667). Other 3′ UTR₅ describedin the mRNA constructs in the non-patent literature include CYBA (Feriziet al., 2015) and albumin (Thess et al., 2015). Other exemplary 3′ UTR₅include that of bovine or human growth hormone (wild type or modified)(WO2013/185069, US20140206753, WO2014152774), rabbit β globin andhepatitis B virus (HBV), β-globin 3′ UTR and Viral VEEV 3′ UTR sequencesare also known in the art. In some embodiments, the sequence UUUGAAUU(WO2014144196) is used. In some embodiments, 3′ UTR₅ of human and mouseribosomal protein are used. Other examples include rps9 3′UTR(WO2015101414), FIG. 4 (WO2015101415), and human albumin 7(WO2015101415).

In some embodiments, a 3′ UTR of the present disclosure comprises asequence selected from SEQ ID NO: 5 and SEQ ID NO: 6.

Those of ordinary skill in the art will understand that 5′UTR₅ that areheterologous or synthetic may be used with any desired 3′ UTR sequence.For example, a heterologous 5′UTR may be used with a synthetic 3′UTRwith a heterologous 3″ UTR.

Non-UTR sequences may also be used as regions or subregions within anucleic acid. For example, introns or portions of introns sequences maybe incorporated into regions of nucleic acid of the disclosure.Incorporation of intronic sequences may increase protein production aswell as nucleic acid levels.

Combinations of features may be included in flanking regions and may becontained within other features. For example, the ORF may be flanked bya 5′ UTR which may contain a strong Kozak translational initiationsignal and/or a 3′ UTR which may include an oligo(dT) sequence fortemplated addition of a poly-A tail. 5′ UTR may comprise a firstpolynucleotide fragment and a second polynucleotide fragment from thesame and/or different genes such as the 5′ UTR₅ described in US PatentApplication Publication No.20100293625 and PCT/US2014/069155, hereinincorporated by reference in its entirety.

It should be understood that any UTR from any gene may be incorporatedinto the regions of a nucleic acid. Furthermore, multiple wild-type UTR₅of any known gene may be utilized. It is also within the scope of thepresent disclosure to provide artificial UTR₅ which are not variants ofwild type regions. These UTR₅ or portions thereof may be placed in thesame orientation as in the transcript from which they were selected ormay be altered in orientation or location. Hence a 5′ or 3′ UTR may beinverted, shortened, lengthened, made with one or more other 5′ UTR₅ or3′ UTRs. As used herein, the term “altered” as it relates to a UTRsequence, means that the UTR has been changed in some way in relation toa reference sequence. For example, a 3′ UTR or 5′ UTR may be alteredrelative to a wild-type or native UTR by the change in orientation orlocation as taught above or may be altered by the inclusion ofadditional nucleotides, deletion of nucleotides, swapping ortransposition of nucleotides. Any of these changes producing an“altered” UTR (whether 3′ or 5′) comprise a variant UTR.

In some embodiments, a double, triple or quadruple UTR such as a 5′ UTRor 3′ UTR may be used. As used herein, a “double” UTR is one in whichtwo copies of the same UTR are encoded either in series or substantiallyin series. For example, a double beta-globin 3′ UTR may be used asdescribed in US Patent publication 20100129877, the contents of whichare incorporated herein by reference in its entirety.

It is also within the scope of the present disclosure to have patternedUTRs. As used herein “patterned UTRs” are those UTR₅ which reflect arepeating or alternating pattern, such as ABABAB or AABBAABBAABB orABCABCABC or variants thereof repeated once, twice, or more than 3times. In these patterns, each letter, A, B, or C represent a differentUTR at the nucleotide level.

In some embodiments, flanking regions are selected from a family oftranscripts whose proteins share a common function, structure, featureor property. For example, polypeptides of interest may belong to afamily of proteins which are expressed in a particular cell, tissue orat some time during development. The UTR₅ from any of these genes may beswapped for any other UTR of the same or different family of proteins tocreate a new polynucleotide. As used herein, a “family of proteins” isused in the broadest sense to refer to a group of two or morepolypeptides of interest which share at least one function, structure,feature, localization, origin, or expression pattern.

The untranslated region may also include translation enhancer elements(TEE). As a non-limiting example, the TEE may include those described inUS Application No.20090226470, herein incorporated by reference in itsentirety, and those known in the art.

In Vitro Transcription of RNA

cDNA encoding the polynucleotides described herein may be transcribedusing an in vitro transcription (IVT) system. In vitro transcription ofRNA is known in the art and is described in International PublicationWO/2014/152027, which is incorporated by reference herein in itsentirety.

In some embodiments, the RNA transcript is generated using anon-amplified, linearized DNA template in an in vitro transcriptionreaction to generate the RNA transcript. In some embodiments, thetemplate DNA is isolated DNA. In some embodiments, the template DNA iscDNA. In some embodiments, the cDNA is formed by reverse transcriptionof a RNA polynucleotide, for example, but not limited to hMPV/hPIV3mRNA. In some embodiments, cells, e.g., bacterial cells, e.g., E. coli,e.g., DH-1 cells are transfected with the plasmid DNA template. In someembodiments, the transfected cells are cultured to replicate the plasmidDNA which is then isolated and purified. In some embodiments, the DNAtemplate includes a RNA polymerase promoter, e.g., a T7 promoter located5′ to and operably linked to the gene of interest.

In some embodiments, an in vitro transcription template encodes a 5′untranslated (UTR) region, contains an open reading frame, and encodes a3′ UTR and a polyA tail. The particular nucleic acid sequencecomposition and length of an in vitro transcription template will dependon the mRNA encoded by the template.

A “5′ untranslated region” (UTR) refers to a region of an mRNA that isdirectly upstream (i.e., 5′) from the start codon (i.e., the first codonof an mRNA transcript translated by a ribosome) that does not encode apolypeptide. When RNA transcripts are being generated, the 5′ UTR maycomprise a promoter sequence. Such promoter sequences are known in theart. It should be understood that such promoter sequences will not bepresent in a vaccine of the disclosure.

A “3′ untranslated region” (UTR) refers to a region of an mRNA that isdirectly downstream (i.e., 3′) from the stop codon (i.e., the codon ofan mRNA transcript that signals a termination of translation) that doesnot encode a polypeptide.

An “open reading frame” is a continuous stretch of DNA beginning with astart codon (e.g., methionine (ATG)), and ending with a stop codon(e.g., TAA, TAG or TGA) and encodes a polypeptide.

A “polyA tail” is a region of mRNA that is downstream, e.g., directlydownstream (i.e., 3′), from the 3′ UTR that contains multiple,consecutive adenosine monophosphates. A polyA tail may contain 10 to 300adenosine monophosphates. For example, a polyA tail may contain 10, 20,30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180,190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300 adenosinemonophosphates. In some embodiments, a polyA tail contains 50 to 250adenosine monophosphates. In a relevant biological setting (e.g., incells, in vivo) the poly(A) tail functions to protect mRNA fromenzymatic degradation, e.g., in the cytoplasm, and aids in transcriptiontermination, and/or export of the mRNA from the nucleus and translation.

In some embodiments, a nucleic acid includes 200 to 3,000 nucleotides.For example, a nucleic acid may include 200 to 500, 200 to 1000, 200 to1500, 200 to 3000, 500 to 1000, 500 to 1500, 500 to 2000, 500 to 3000,1000 to 1500, 1000 to 2000, 1000 to 3000, 1500 to 3000, or 2000 to 3000nucleotides).

An in vitro transcription system typically comprises a transcriptionbuffer, nucleotide triphosphates (NTPs), an RNase inhibitor and apolymerase.

The NTPs may be manufactured in house, may be selected from a supplier,or may be synthesized as described herein. The NTPs may be selectedfrom, but are not limited to, those described herein including naturaland unnatural (modified) NTPs.

Any number of RNA polymerases or variants may be used in the method ofthe present disclosure. The polymerase may be selected from, but is notlimited to, a phage RNA polymerase, e.g., a T7 RNA polymerase, a T3 RNApolymerase, a SP6 RNA polymerase, and/or mutant polymerases such as, butnot limited to, polymerases able to incorporate modified nucleic acidsand/or modified nucleotides, including chemically modified nucleic acidsand/or nucleotides. Some embodiments exclude the use of DNase.

In some embodiments, the RNA transcript is capped via enzymatic capping.In some embodiments, the RNA comprises 5′ terminal cap, for example,7mG(5′)ppp(5′)NlmpNp.

Chemical Synthesis

Solid-phase chemical synthesis. Nucleic acids the present disclosure maybe manufactured in whole or in part using solid phase techniques.Solid-phase chemical synthesis of nucleic acids is an automated methodwherein molecules are immobilized on a solid support and synthesizedstep by step in a reactant solution. Solid-phase synthesis is useful insite-specific introduction of chemical modifications in the nucleic acidsequences.

Liquid Phase Chemical Synthesis. The synthesis of nucleic acids of thepresent disclosure by the sequential addition of monomer building blocksmay be carried out in a liquid phase.

Combination of Synthetic Methods. The synthetic methods discussed aboveeach has its own advantages and limitations. Attempts have beenconducted to combine these methods to overcome the limitations. Suchcombinations of methods are within the scope of the present disclosure.The use of solid-phase or liquid-phase chemical synthesis in combinationwith enzymatic ligation provides an efficient way to generate long chainnucleic acids that cannot be obtained by chemical synthesis alone.

Ligation of Nucleic Acid Regions or Subregions

Assembling nucleic acids by a ligase may also be used. DNA or RNAligases promote intermolecular ligation of the 5′ and 3′ ends ofpolynucleotide chains through the formation of a phosphodiester bond.Nucleic acids such as chimeric polynucleotides and/or circular nucleicacids may be prepared by ligation of one or more regions or subregions.DNA fragments can be joined by a ligase catalyzed reaction to createrecombinant DNA with different functions. Two oligodeoxynucleotides, onewith a 5′ phosphoryl group and another with a free 3′ hydroxyl group,serve as substrates for a DNA ligase.

Purification

Purification of the nucleic acids described herein may include, but isnot limited to, nucleic acid clean-up, quality assurance and qualitycontrol. Clean-up may be performed by methods known in the arts such as,but not limited to, AGENCOURT® beads (Beckman Coulter Genomics, Danvers,Mass.), poly-T beads, LNATM oligo-T capture probes (EXIQON® Inc,Vedbaek, Denmark) or HPLC based purification methods such as, but notlimited to, strong anion exchange HPLC, weak anion exchange HPLC,reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC(HIC-HPLC). The term “purified” when used in relation to a nucleic acidsuch as a “purified nucleic acid” refers to one that is separated fromat least one contaminant. A “contaminant” is any substance that makesanother unfit, impure or inferior. Thus, a purified nucleic acid (e.g.,DNA and RNA) is present in a form or setting different from that inwhich it is found in nature, or a form or setting different from thatwhich existed prior to subjecting it to a treatment or purificationmethod.

A quality assurance and/or quality control check may be conducted usingmethods such as, but not limited to, gel electrophoresis, UV absorbance,or analytical HPLC.

In some embodiments, the nucleic acids may be sequenced by methodsincluding, but not limited to reverse-transcriptase-PCR.

Quantification

In some embodiments, the nucleic acids of the present disclosure may bequantified in exosomes or when derived from one or more bodily fluid.Bodily fluids include peripheral blood, serum, plasma, ascites, urine,cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid,aqueous humor, amniotic fluid, cerumen, breast milk, broncheoalveolarlavage fluid, semen, prostatic fluid, cowper's fluid or pre-ejaculatoryfluid, sweat, fecal matter, hair, tears, cyst fluid, pleural andperitoneal fluid, pericardial fluid, lymph, chyme, chyle, bile,interstitial fluid, menses, pus, sebum, vomit, vaginal secretions,mucosal secretion, stool water, pancreatic juice, lavage fluids fromsinus cavities, bronchopulmonary aspirates, blastocyl cavity fluid, andumbilical cord blood. Alternatively, exosomes may be retrieved from anorgan selected from the group consisting of lung, heart, pancreas,stomach, intestine, bladder, kidney, ovary, testis, skin, colon, breast,prostate, brain, esophagus, liver, and placenta.

Assays may be performed using construct specific probes, cytometry,qRT-PCR, real-time PCR, PCR, flow cytometry, electrophoresis, massspectrometry, or combinations thereof while the exosomes may be isolatedusing immunohistochemical methods such as enzyme linked immunosorbentassay (ELISA) methods. Exosomes may also be isolated by size exclusionchromatography, density gradient centrifugation, differentialcentrifugation, nanomembrane ultrafiltration, immunoabsorbent capture,affinity purification, microfluidic separation, or combinations thereof.

These methods afford the investigator the ability to monitor, in realtime, the level of nucleic acids remaining or delivered. This ispossible because the nucleic acids of the present disclosure, in someembodiments, differ from the endogenous forms due to the structural orchemical modifications.

In some embodiments, the nucleic acid may be quantified using methodssuch as, but not limited to, ultraviolet visible spectroscopy (UV/Vis).A non-limiting example of a UV/Vis spectrometer is a NANODROP®spectrometer (ThermoFisher, Waltham, Mass.). The quantified nucleic acidmay be analyzed in order to determine if the nucleic acid may be ofproper size, check that no degradation of the nucleic acid has occurred.Degradation of the nucleic acid may be checked by methods such as, butnot limited to, agarose gel electrophoresis, HPLC based purificationmethods such as, but not limited to, strong anion exchange HPLC, weakanion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobicinteraction HPLC (HIC-HPLC), liquid chromatography-mass spectrometry(LCMS), capillary electrophoresis (CE) and capillary gel electrophoresis(CGE).

Lipid Nanoparticles (LNPs)

In some embodiments, the hMPV/hPIV3 mRNA vaccine of the disclosure isformulated in a lipid nanoparticle (LNP). Lipid nanoparticles typicallycomprise ionizable cationic lipid, non-cationic lipid, sterol and PEGlipid components along with the nucleic acid cargo of interest. Thelipid nanoparticles of the disclosure can be generated using components,compositions, and methods as are generally known in the art, see forexample PCT/US2016/052352; PCT/US2016/068300; PCT/US2017/037551;PCT/US2015/027400; PCT/US2016/047406; PCT/US2016000129;PCT/US2016/014280; PCT/US2016/014280; PCT/US2017/038426;PCT/US2014/027077; PCT/US2014/055394; PCT/US2016/52117;PCT/US2012/069610; PCT/US2017/027492; PCT/US2016/059575 andPCT/US2016/069491 all of which are incorporated by reference herein intheir entirety.

Vaccines of the present disclosure are typically formulated in lipidnanoparticle. In some embodiments, the lipid nanoparticle comprises atleast one ionizable cationic lipid, at least one non-cationic lipid, atleast one sterol, and/or at least one polyethylene glycol (PEG)-modifiedlipid.

In some embodiments, the lipid nanoparticle comprises a molar ratio of20-60% ionizable cationic lipid. For example, the lipid nanoparticle maycomprise a molar ratio of 20-50%, 20-40%, 20-30%, 30-60%, 30-50%,30-40%, 40-60%, 40-50%, or 50-60% ionizable cationic lipid. In someembodiments, the lipid nanoparticle comprises a molar ratio of 20%, 30%,40%, 50, or 60% ionizable cationic lipid.

In some embodiments, the lipid nanoparticle comprises a molar ratio of5-25% non-cationic lipid. For example, the lipid nanoparticle maycomprise a molar ratio of 5-20%, 5-15%, 5-10%, 10-25%, 10-20%, 10-25%,15-25%, 15-20%, or 20-25% non-cationic lipid. In some embodiments, thelipid nanoparticle comprises a molar ratio of 5%, 10%, 15%, 20%, or25%non-cationic lipid.

In some embodiments, the lipid nanoparticle comprises a molar ratio of25-55% sterol. For example, the lipid nanoparticle may comprise a molarratio of 25-50%, 25-45%, 25-40%, 25-35%, 25-30%, 30-55%, 30-50%, 30-45%,30-40%, 30-35%, 35-55%, 35-50%, 35-45%, 35-40%, 40-55%, 40-50%, 40-45%,45-55%, 45-50%, or 50-55% sterol. In some embodiments, the lipidnanoparticle comprises a molar ratio of 25%, 30%, 35%, 40%, 45%, 50%, or55% sterol.

In some embodiments, the lipid nanoparticle comprises a molar ratio of0.5-15% PEG-modified lipid. For example, the lipid nanoparticle maycomprise a molar ratio of 0.5-10%, 0.5-5%, 1-15%, 1-10%, 1-5%, 2-15%,2-10%, 2-5%, 5-15%, 5-10%, or 10-15%. In some embodiments, the lipidnanoparticle comprises a molar ratio of 0.5%, 1%, 2%, 3%, 4%, 5%, 6%,7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% PEG-modified lipid.

In some embodiments, the lipid nanoparticle comprises a molar ratio of20-60% ionizable cationic lipid, 5-25% non-cationic lipid, 25-55%sterol, and 0.5-15% PEG-modified lipid.

In some embodiments, an ionizable cationic lipid of the disclosurecomprises a compound of Formula (I):

or a salt or isomer thereof, wherein:

R₁ is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀ alkenyl,—R*YR″, —YR″, and —R″M′R′;

R₂ and R₃ are independently selected from the group consisting of H,C₁₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or R₂ and R₃,together with the atom to which they are attached, form a heterocycle orcarbocycle;

R₄ is selected from the group consisting of a C₃₋₆ carbocycle,—(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, —CQ(R)₂, and unsubstituted C₁₋₆alkyl, where Q is selected from a carbocycle, heterocycle, —OR,—O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃, —CX₂H, —CXH₂, —CN, —N(R)₂,—C(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R, —N(R)C(O)N(R)₂, —N(R)C(S)N(R)₂,—N(R)R₈, —O(CH₂)nOR, —N(R)C(═NR₉)N(R)₂, —N(R)C(═CHR₉)N(R)₂, —OC(O)N(R)₂,—N(R)C(O)OR, —N(OR)C(O)R, —N(OR)S(O)₂R, —N(OR)C(O)OR, —N(OR)C(O)N(R)₂,—N(OR)C(S)N(R)₂, —N(OR)C(═NR₉)N(R)₂, —N(OR)C(═CHR₉)N(R)₂, —C(═NR₉)N(R)₂,—C(═NR₉)R, —C(O)N(R)OR, and —C(R)N(R)₂C(O)OR, and each n isindependently selected from 1, 2, 3, 4, and 5;

each R₅ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R₆ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—,

—N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—,—S(O)₂—, —S—S—, an aryl group, and a heteroaryl group;

R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl,and H;

R₈ is selected from the group consisting of C₃₋₆ carbocycle andheterocycle;

R₉ is selected from the group consisting of H, CN, NO₂, C₁₋₆ alkyl, —OR,—S(O)₂R, —S(O)₂N(R)₂, C₂₋₆ alkenyl, C₃₋₆ carbocycle and heterocycle;

each R is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R′ is independently selected from the group consisting of C₁₋₁₈alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;

each R″ is independently selected from the group consisting of C₃₋₁₄alkyl and C₃₋₁₄ alkenyl;

each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₂₋₁₂ alkenyl;

each Y is independently a C₃₋₆ carbocycle;

each X is independently selected from the group consisting of F, Cl, Br,and I; and

m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13.

In some embodiments, a subset of compounds of Formula (I) includes thosein which when R₄ is —(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, or —CQ(R)₂, then(i) Q is not —N(R)₂ when n is 1, 2, 3, 4 or 5, or (ii) Q is not 5, 6, or7-membered heterocycloalkyl when n is 1 or 2.

In some embodiments, another subset of compounds of Formula (I) includesthose in which

R₁ is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀ alkenyl,—R*YR″, —YR″, and —R″M′R′;

R₂ and R₃ are independently selected from the group consisting of H,C₁₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or R₂ and R₃,together with the atom to which they are attached, form a heterocycle orcarbocycle;

R₄ is selected from the group consisting of a C₃₋₆ carbocycle,—(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, —CQ(R)₂, and unsubstituted C₁₋₆alkyl, where Q is selected from a C₃₋₆ carbocycle, a 5-to 14-memberedheteroaryl having one or more heteroatoms selected from N, O, and S,—OR, —O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃, —CX₂H, —CXH₂, —CN,—C(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R, —N(R)C(O)N(R)₂, —N(R)C(S)N(R)₂,—CRN(R)₂C(O)OR, —N(R)R₈, —O(CH₂)_(n)OR, —N(R)C(═NR₉)N(R)₂,—N(R)C(═CHR₉)N(R)₂, —OC(O)N(R)₂, —N(R)C(O)OR, —N(OR)C(O)R, —N(OR)S(O)₂R,—N(OR)C(O)OR, —N(OR)C(O)N(R)₂, —N(OR)C(S)N(R)₂, —N(OR)C(═NR₉)N(R)₂,—N(OR)C(═CHR₉)N(R)₂, —C(═NR₉)N(R)₂, —C(═NR₉)R, —C(O)N(R)OR, and a 5- to14-membered heterocycloalkyl having one or more heteroatoms selectedfrom N, O, and S which is substituted with one or more substituentsselected from oxo (═O), OH, amino, mono- or di-alkylamino, and C₁₋₃alkyl, and each n is independently selected from 1, 2, 3, 4, and 5;

each R₅ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R₆ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—,—N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—,—S(O)₂—, —S—S—, an aryl group, and a heteroaryl group;

R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl,and H;

R₈ is selected from the group consisting of C₃₋₆ carbocycle andheterocycle;

R₉ is selected from the group consisting of H, CN, NO₂, C₁₋₆ alkyl, —OR,—S(O)₂R, —S(O)₂N(R)₂, C₂₋₆ alkenyl, C₃₋₆ carbocycle and heterocycle;

each R is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R′ is independently selected from the group consisting of C₁₋₁₈alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;

each R″ is independently selected from the group consisting of C₃₋₁₄alkyl and C₃₋₁₄ alkenyl;

each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₂₋₁₂ alkenyl;

each Y is independently a C₃₋₆ carbocycle;

each X is independently selected from the group consisting of F, Cl, Br,and I; and

m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,

or salts or isomers thereof.

In some embodiments, another subset of compounds of Formula (I) includesthose in which

R₁ is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀ alkenyl,—R*YR″, —YR″, and —R″M′R′;

R₂ and R₃ are independently selected from the group consisting of H,C₁₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or R₂ and R₃,together with the atom to which they are attached, form a heterocycle orcarbocycle;

R₄ is selected from the group consisting of a C₃₋₆ carbocycle,—(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, —CQ(R)₂, and unsubstituted C₁₋₆alkyl, where Q is selected from a C₃₋₆ carbocycle, a 5-to 14-memberedheterocycle having one or more heteroatoms selected from N, O, and S,—OR, —O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃, —CX₂H, —CXH₂, —CN,—C(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R, —N(R)C(O)N(R)₂, —N(R)C(S)N(R)₂,—CRN(R)₂C(O)OR, —N(R)R₈, —O(CH₂)_(n)OR, —N(R)C(═NR₉)N(R)₂,—N(R)C(═CHR₉)N(R)₂, —OC(O)N(R)₂, —N(R)C(O) OR, —N(OR)C(O)R,—N(OR)S(O)₂R, —N(OR)C(O)OR, —N(OR)C(O)N(R)₂, —N(OR)C(S)N(R)₂, —N(OR)C(═NR₉)N(R)₂, —N(OR)C(═CHR₉)N(R)₂, —C(═NR₉)R, —C(O)N(R)OR, and—C(═NR₉)N(R)₂, and each n is independently selected from 1, 2, 3, 4, and5; and when Q is a 5- to 14-membered heterocycle and (i) R₄ is—(CH₂)_(n)Q in which n is 1 or 2, or (ii) R₄ is —(CH₂)_(n)CHQR in whichn is 1, or (iii) R₄ is —CHQR, and —CQ(R)₂, then Q is either a 5- to14-membered heteroaryl or 8- to 14-membered heterocycloalkyl;

each R₅ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R₆ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—,—N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—,—S(O)₂—, —S—S—, an aryl group, and a heteroaryl group;

R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl,and H;

R₈ is selected from the group consisting of C₃₋₆ carbocycle andheterocycle;

R₉ is selected from the group consisting of H, CN, NO₂, C₁₋₆ alkyl, —OR,—S(O)₂R, —S(O)₂N(R)₂, C₂₋₆ alkenyl, C₃₋₆ carbocycle and heterocycle;

each R is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R′ is independently selected from the group consisting of C₁₋₁₈alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;

each R″ is independently selected from the group consisting of C₃₋₁₄alkyl and C₃₋₁₄ alkenyl;

each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₂₋₁₂ alkenyl;

each Y is independently a C₃₋₆ carbocycle;

each X is independently selected from the group consisting of F, Cl, Br,and I; and

m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,

or salts or isomers thereof.

In some embodiments, another subset of compounds of Formula (I) includesthose in which

R₁ is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀ alkenyl,—R*YR″, —YR″, and —R″M′R′;

R₂ and R₃ are independently selected from the group consisting of H,C₁₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or R₂ and R₃,together with the atom to which they are attached, form a heterocycle orcarbocycle;

R₄ is selected from the group consisting of a C₃₋₆ carbocycle,—(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, —CQ(R)₂, and unsubstituted C₁₋₆alkyl, where Q is selected from a C₃₋₆ carbocycle, a 5- to 14-memberedheteroaryl having one or more heteroatoms selected from N, O, and S,—OR, —O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃, —CX₂H, —CXH₂, —CN,—C(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R, —N(R)C(O)N(R)₂, —N(R)C(S)N(R)₂,—CRN(R)₂C(O)OR, —N(R)R₈, —O(CH₂)_(n)OR, —N(R)C(═NR₉)N(R)₂,—N(R)C(═CHR₉)N(R)₂, —OC(O)N(R)₂, —N(R)C(O)OR, —N(OR)C(O)R, —N(OR)S(O)₂R,—N(OR)C(O)OR, —N(OR)C(O)N(R)₂, —N(OR)C(S)N(R)₂, —N(OR)C(═NR₉)N(R)₂,—N(OR)C(═CHR₉)N(R)₂, —C(═NR₉)R, —C(O)N(R)OR, and —C(═NR₉)N(R)₂, and eachn is independently selected from 1, 2, 3, 4, and 5;

each R₅ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R₆ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—,—N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—,—S(O)₂—, —S—S—, an aryl group, and a heteroaryl group;

R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl,and H;

R₈ is selected from the group consisting of C₃₋₆ carbocycle andheterocycle;

R₉ is selected from the group consisting of H, CN, NO₂, C₁₋₆ alkyl, —OR,—S(O)₂R, —S(O)₂N(R)₂, C₂₋₆ alkenyl, C₃₋₆ carbocycle and heterocycle;

each R is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R′ is independently selected from the group consisting of C₁₋₁₈alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;

each R″ is independently selected from the group consisting of C₃₋₁₄alkyl and C₃₋₁₄ alkenyl;

each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₂₋₁₂ alkenyl;

each Y is independently a C₃₋₆ carbocycle;

each X is independently selected from the group consisting of F, Cl, Br,and I; and

m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,

or salts or isomers thereof.

In some embodiments, another subset of compounds of Formula (I) includesthose in which

R₁ is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀ alkenyl,—R*YR″, —YR″, and —R″M′R′;

R₂ and R₃ are independently selected from the group consisting of H,C₂₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or R₂ and R₃,together with the atom to which they are attached, form a heterocycle orcarbocycle;

R₄ is —(CH₂)_(n)Q or —(CH₂)_(n)CHQR, where Q is —N(R)₂, and n isselected from 3, 4, and 5;

each R₅ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R₆ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—,—N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—,—S(O)₂—, —S—S—, an aryl group, and a heteroaryl group;

R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl,and H;

each R is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R′ is independently selected from the group consisting of C₁₋₁₈alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;

each R″ is independently selected from the group consisting of C₃₋₁₄alkyl and C₃₋₁₄ alkenyl;

each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₁₋₁₂

each Y is independently a C₃₋₆ carbocycle;

each X is independently selected from the group consisting of F, Cl, Br,and I; and

m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,

or salts or isomers thereof.

In some embodiments, another subset of compounds of Formula (I) includesthose in which

R₁ is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀ alkenyl,—R*YR″, —YR″, and —R″M′R′;

R₂ and R₃ are independently selected from the group consisting of C₁₋₁₄alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or R₂ and R₃, togetherwith the atom to which they are attached, form a heterocycle orcarbocycle;

R₄ is selected from the group consisting of —(CH₂)_(n)Q, —(CH₂)_(n)CHQR,—CHQR, and —CQ(R)₂, where Q is —N(R)₂, and n is selected from 1, 2, 3,4, and 5;

each R₅ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R₆ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—,—N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—,—S(O)₂—, —S—S—, an aryl group, and a heteroaryl group;

R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl,and H;

each R is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R′ is independently selected from the group consisting of C₁₋₁₈alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;

each R″ is independently selected from the group consisting of C₃₋₁₄alkyl and C₃₋₁₄ alkenyl;

each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₁₋₁₂ alkenyl;

each Y is independently a C₃₋₆ carbocycle;

each X is independently selected from the group consisting of F, Cl, Br,and I; and

m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,

or salts or isomers thereof.

In some embodiments, a subset of compounds of Formula (I) includes thoseof Formula (IA):

or a salt or isomer thereof, wherein 1 is selected from 1, 2, 3, 4, and5; m is selected from 5, 6, 7, 8, and 9; M₁ is a bond or M′; R₄ isunsubstituted C₁₋₃ alkyl, or —(CH₂)_(n)Q, in which Q is OH,—NHC(S)N(R)₂, —NHC(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R, —N(R)R₈,—NHC(═NR₉)N(R)₂, —NHC(═CHR₉)N(R)₂, —OC(O)N(R)₂, —N(R)C(O)OR, heteroarylor heterocycloalkyl; M and M′ are independently selected from —C(O)O—,—OC(O)—, —C(O)N(R′)—, —P(O)(OR′)O—, —S—S—, an aryl group, and aheteroaryl group; and R₂ and R₃ are independently selected from thegroup consisting of H, C₁₋₄ alkyl, and C₂₋₁₄ alkenyl.

In some embodiments, a subset of compounds of Formula (I) includes thoseof Formula

or a salt or isomer thereof, wherein 1 is selected from 1, 2, 3, 4, and5; M₁ is a bond or M′; R₄ is unsubstituted C₁₋₃ alkyl, or —(CH₂)_(n)Q,in which n is 2, 3, or 4, and Q is OH, —NHC(S)N(R)₂, —NHC(O)N(R)₂,—N(R)C(O)R, —N(R)S(O)₂R, —N(R)R₈, —NHC(═NR₉)N(R)₂, —NHC(═CHR₉)N(R)₂,—OC(O)N(R)₂, —N(R)C(O)OR, heteroaryl or heterocycloalkyl; M and M′ areindependently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—, —P(O)(OR′)O—,—S—S—, an aryl group, and a heteroaryl group; and R₂ and R₃ areindependently selected from the group consisting of H, C₁₋₄ alkyl, andC₂₋₁₄ alkenyl.

In some embodiments, a subset of compounds of Formula (I) includes thoseof Formula (IIa), (IIb), (IIc), or (IIe):

or a salt or isomer thereof, wherein R₄ is as described herein.

In some embodiments, a subset of compounds of Formula (I) includes thoseof Formula (IId):

or a salt or isomer thereof, wherein n is 2, 3, or 4; and m, R′, R″, andR₂ through R₆ are as described herein. For example, each of R₂ and R₃may be independently selected from the group consisting of C₅₋₁₄ alkyland C₅₋₁₄ alkenyl.

In some embodiments, an ionizable cationic lipid of the disclosurecomprises a compound having structure:

In some embodiments, an ionizable cationic lipid of the disclosurecomprises a compound having structure:

In some embodiments, a non-cationic lipid of the disclosure comprises1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC),1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE),1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC),1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC),1,2-dioleoyl-sn-glycero-3-phosphocholine(DOPC),1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC),1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC),1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC),1-oleoyl-2 cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine(OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC),1,2-dilinolenoyl-sn-glycero-3-phosphocholine,1,2-diarachidonoyl-sn-glycero-3-phosphocholine,1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine,1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE),1,2-distearoyl-sn-glycero-3-phosphoethanolamine,1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine,1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine,1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine,1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine,1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG),sphingomyelin, and mixtures thereof.

In some embodiments, a PEG modified lipid of the disclosure comprises aPEG-modified phosphatidylethanolamine, a PEG-modified phosphatidic acid,a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG-modifieddiacylglycerol, a PEG-modified dialkylglycerol, and mixtures thereof. Insome embodiments, the PEG-modified lipid is DMG-PEG, PEG-c-DOMG (alsoreferred to as PEG-DOMG), PEG-DSG and/or PEG-DPG.

In some embodiments, a sterol of the disclosure comprises cholesterol,fecosterol, sitosterol, ergosterol, campesterol, stigmasterol,brassicasterol, tomatidine, ursolic acid, alpha-tocopherol, and mixturesthereof.

In some embodiments, a LNP of the disclosure comprises an ionizablecationic lipid of Compound 1, wherein the non-cationic lipid is DSPC,the structural lipid that is cholesterol, and the PEG lipid is DMG-PEG.

In some embodiments, the lipid nanoparticle comprises 45-55 mole percentionizable cationic lipid. For example, lipid nanoparticle may comprise45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 mole percent ionizablecationic lipid.

In some embodiments, the lipid nanoparticle comprises 5-15 mole percentDSPC. For example, the lipid nanoparticle may comprise 5, 6, 7, 8, 9,10, 11, 12, 13, 14, or 15 mole percent DSPC.

In some embodiments, the lipid nanoparticle comprises 35-40 mole percentcholesterol. For example, the lipid nanoparticle may comprise 35, 36,37, 38, 39, or 40 mole percent cholesterol.

In some embodiments, the lipid nanoparticle comprises 1-2 mole percentDMG-PEG. For example, the lipid nanoparticle may comprise 1, 1.5, or 2mole percent DMG-PEG.

In some embodiments, the lipid nanoparticle comprises 50 mole percentionizable cationic lipid, 10 mole percent DSPC, 38.5 mole percentcholesterol, and 1.5 mole percent DMG-PEG.

In some embodiments, a LNP of the disclosure comprises an N:P ratio offrom about 2:1 to about 30:1.

In some embodiments, a LNP of the disclosure comprises an N:P ratio ofabout 6:1.

In some embodiments, a LNP of the disclosure comprises an N:P ratio ofabout 3:1.

In some embodiments, a LNP of the disclosure comprises a wt/wt ratio ofthe ionizable cationic lipid component to the RNA of from about 10:1 toabout 100:1.

In some embodiments, a LNP of the disclosure comprises a wt/wt ratio ofthe ionizable cationic lipid component to the RNA of about 20:1.

In some embodiments, a LNP of the disclosure comprises a wt/wt ratio ofthe ionizable cationic lipid component to the RNA of about 10:1.

In some embodiments, a LNP of the disclosure has a mean diameter fromabout 50 nm to about 150 nm.

In some embodiments, a LNP of the disclosure has a mean diameter fromabout 70 nm to about 120 nm.

Multivalent Vaccines

The hMPV/hPIV3 vaccines, as provided herein, may include mRNA ormultiple mRNAs encoding two or more antigens of the same or differenthMPV/hPIV3 species. In some embodiments, the hMPV/hPIV3 vaccine includesan RNA or multiple RNAs encoding two or more antigens. In someembodiments, the mRNA of a hMPV/hPIV3 vaccine may encode 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, or more antigens.

In some embodiments, the hMPV/hPIV3 mRNA vaccine comprises at least oneRNA encoding a hMPV F glycoprotein and a hPIV3 F glycoprotein antigen.

In some embodiments, two or more different RNA (e.g., mRNA) encodingantigens may be formulated in the same lipid nanoparticle. In otherembodiments, two or more different RNA encoding antigens may beformulated in separate lipid nanoparticles (each RNA formulated in asingle lipid nanoparticle). The lipid nanoparticles may then be combinedand administered as a single vaccine composition (e.g., comprisingmultiple RNA encoding multiple antigens) or may be administeredseparately.

Combination Vaccines

The hMPV/hPIV3 mRNA vaccines, as provided herein, may include an RNA ormultiple RNAs encoding two or more antigens of the same or differenthMPV/hPIV3 strains. Also provided herein are combination vaccines thatinclude RNA encoding one or more hMPV/hPIV3 antigen(s) and one or moreantigen(s) of a different organisms. Thus, the vaccines of the presentdisclosure may be combination vaccines that target one or more antigensof the same strain/species, or one or more antigens of differentstrains/species, e.g., antigens which induce immunity to organisms whichare found in the same geographic areas where the risk of hMPV/hPIV3infection is high or organisms to which an individual is likely to beexposed to when exposed to hMPV/hPIV3.

Pharmaceutical Formulations

Provided herein are compositions (e.g., pharmaceutical compositions),methods, kits and reagents for prevention or treatment of hMPV/hPIV3 inhumans and other mammals, for example. hMPV/hPIV3 mRNA vaccines can beused as therapeutic or prophylactic agents. They may be used in medicineto prevent and/or treat infectious disease.

In some embodiments, the hMPV/hPIV3 vaccine containing mRNA as describedherein can be administered to a subject (e.g., a mammalian subject, suchas a human subject), and the RNA polynucleotides are translated in vivoto produce an antigenic polypeptide (antigen).

An “effective amount” of a hMPV/hPIV3 vaccine is based, at least inpart, on the target tissue, target cell type, means of administration,physical characteristics of the RNA (e.g., length, nucleotidecomposition, and/or extent of modified nucleosides), other components ofthe vaccine, and other determinants, such as age, body weight, height,sex and general health of the subject. Typically, an effective amount ofa hMPV/hPIV3 mRNA vaccine provides an induced or boosted immune responseas a function of antigen production in the cells of the subject. In someembodiments, an effective amount of the hMPV/hPIV3 mRNA vaccinecontaining RNA polynucleotides having at least one chemicalmodifications are more efficient than a composition containing acorresponding unmodified polynucleotide encoding the same antigen or apeptide antigen. Increased antigen production may be demonstrated byincreased cell transfection (the percentage of cells transfected withthe RNA vaccine), increased protein translation and/or expression fromthe polynucleotide, decreased nucleic acid degradation (as demonstrated,for example, by increased duration of protein translation from amodified polynucleotide), or altered antigen specific immune response ofthe host cell.

The term “pharmaceutical composition” refers to the combination of anactive agent with a carrier, inert or active, making the compositionespecially suitable for diagnostic or therapeutic use in vivo or exvivo. A “pharmaceutically acceptable carrier,” after administered to orupon a subject, does not cause undesirable physiological effects. Thecarrier in the pharmaceutical composition must be “acceptable” also inthe sense that it is compatible with the active ingredient and can becapable of stabilizing it. One or more solubilizing agents can beutilized as pharmaceutical carriers for delivery of an active agent.Examples of a pharmaceutically acceptable carrier include, but are notlimited to, biocompatible vehicles, adjuvants, additives, and diluentsto achieve a composition usable as a dosage form. Examples of othercarriers include colloidal silicon oxide, magnesium stearate, cellulose,and sodium lauryl sulfate. Additional suitable pharmaceutical carriersand diluents, as well as pharmaceutical necessities for their use, aredescribed in Remington's Pharmaceutical Sciences.

In some embodiments, RNA vaccines (including polynucleotides and theirencoded polypeptides) in accordance with the present disclosure may beused for treatment or prevention of hMPV/hPIV3. The hMPV/hPIV3 mRNAvaccine may be administered prophylactically or therapeutically as partof an active immunization scheme to healthy individuals or early ininfection during the incubation phase or during active infection afteronset of symptoms. In some embodiments, the amount of RNA vaccines ofthe present disclosure provided to a cell, a tissue or a subject may bean amount effective for immune prophylaxis.

The hMPV/hPIV3 mRNA vaccine may be administered with other prophylacticor therapeutic compounds. As a non-limiting example, a prophylactic ortherapeutic compound may be an adjuvant or a booster. As used herein,when referring to a prophylactic composition, such as a vaccine, theterm “booster” refers to an extra administration of the prophylactic(vaccine) composition. A booster (or booster vaccine) may be given afteran earlier administration of the prophylactic composition. The time ofadministration between the initial administration of the prophylacticcomposition and the booster may be, but is not limited to, 1 minute, 2minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes 35 minutes, 40minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 2 hours, 3 hours, 4hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours,12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 36 hours, 2 days,3 days, 4 days, 5 days, 6 days, 1 week, 10 days, 2 weeks, 3 weeks, 1month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8months, 9 months, 10 months, 11 months, 1 year, 18 months, 2 years, 3years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years,11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18years, 19 years, 20 years, 25 years, 30 years, 35 years, 40 years, 45years, 50 years, 55 years, 60 years, 65 years, 70 years, 75 years, 80years, 85 years, 90 years, 95 years or more than 99 years. In exemplaryembodiments, the time of administration between the initialadministration of the prophylactic composition and the booster may be,but is not limited to, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3months, 6 months or 1 year.

In some embodiments, the hMPV/hPIV3 mRNA vaccine may be administeredintramuscularly, intranasally or intradermally, similarly to theadministration of inactivated vaccines known in the art.

The hMPV/hPIV3 mRNA vaccine may be utilized in various settingsdepending on the prevalence of the infection or the degree or level ofunmet medical need. As a non-limiting example, the RNA vaccines may beutilized to treat and/or prevent a variety of infectious disease. RNAvaccines have superior properties in that they produce much largerantibody titers, better neutralizing immunity, produce more durableimmune responses, and/or produce responses earlier than commerciallyavailable vaccines.

Provided herein are pharmaceutical compositions including the hMPV/hPIV3mRNA vaccine and/or complexes optionally in combination with one or morepharmaceutically acceptable excipients.

The hMPV/hPIV3 mRNA vaccine may be formulated or administered alone orin conjunction with one or more other components. For instance, thehMPV/hPIV3 mRNA vaccine may comprise other components including, but notlimited to, adjuvants.

In some embodiments, the hMPV/hPIV3 mRNA vaccine does not include anadjuvant (they are adjuvant free).

The hMPV/hPIV3 mRNA vaccine may be formulated or administered incombination with one or more pharmaceutically-acceptable excipients. Insome embodiments, vaccine compositions comprise at least one additionalactive substances, such as, for example, a therapeutically-activesubstance, a prophylactically-active substance, or a combination ofboth. Vaccine compositions may be sterile, pyrogen-free or both sterileand pyrogen-free. General considerations in the formulation and/ormanufacture of pharmaceutical agents, such as vaccine compositions, maybe found, for example, in Remington: The Science and Practice ofPharmacy 21st ed., Lippincott Williams & Wilkins, 2005 (incorporatedherein by reference in its entirety).

In some embodiments, the hMPV/hPIV3 mRNA vaccine are administered tohumans, human patients or subjects. For the purposes of the presentdisclosure, the phrase “active ingredient” generally refers to the RNAvaccines or the polynucleotides contained therein, for example, RNApolynucleotides (e.g., mRNA polynucleotides) encoding antigens.

Formulations of the vaccine compositions described herein may beprepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the active ingredient (e.g., mRNA polynucleotide) intoassociation with an excipient and/or one or more other accessoryingredients, and then, if necessary and/or desirable, dividing, shapingand/or packaging the product into a desired single-or multi-dose unit.

Relative amounts of the active ingredient, the pharmaceuticallyacceptable excipient, and/or any additional ingredients in apharmaceutical composition in accordance with the disclosure will vary,depending upon the identity, size, and/or condition of the subjecttreated and further depending upon the route by which the composition isto be administered. By way of example, the composition may comprisebetween 0.1% and 100%, e.g., between 0.5 and 50%, between 1-30%, between5-80%, at least 80% (w/w) active ingredient.

In some embodiments, the hMPV/hPIV3 mRNA vaccine is formulated using oneor more excipients to: (1) increase stability; (2) increase celltransfection; (3) permit the sustained or delayed release (e.g., from adepot formulation); (4) alter the biodistribution (e.g., target tospecific tissues or cell types); (5) increase the translation of encodedprotein in vivo; and/or (6) alter the release profile of encoded protein(antigen) in vivo. In addition to traditional excipients such as any andall solvents, dispersion media, diluents, or other liquid vehicles,dispersion or suspension aids, surface active agents, isotonic agents,thickening or emulsifying agents, preservatives, excipients can include,without limitation, lipidoids, liposomes, lipid nanoparticles, polymers,lipoplexes, core-shell nanoparticles, peptides, proteins, cellstransfected with the hMPV/hPIV3 mRNA vaccine (e.g., for transplantationinto a subject), hyaluronidase, nanoparticle mimics and combinationsthereof.

Dosing/Administration

Provided herein are compositions (e.g., pharmaceutical compositions),methods, kits and reagents for prevention and/or treatment of hMPV/hPIV3in humans and other mammals. The hMPV/hPIV3 vaccine can be used astherapeutic or prophylactic agents. In some aspects, the RNA vaccines ofthe disclosure are used to provide prophylactic protection fromhMPV/hPIV3. In some aspects, the RNA vaccines of the disclosure are usedto treat a hMPV/hPIV3 infection. In some embodiments, the hMPV/hPIV3mRNA vaccine of the present disclosure is used in the priming of immuneeffector cells, for example, to activate peripheral blood mononuclearcells (PBMCs) ex vivo, which are then infused (re-infused) into asubject.

A subject may be any mammal, including non-human primate and humansubjects. Typically, a subject is a human subject.

In some embodiments, the hMPV/hPIV3 mRNA vaccine is administered to asubject (e.g., a mammalian subject, such as a human subject) in aneffective amount to induce an antigen-specific immune response. The RNAencoding the hMPV/hPIV3 antigen is expressed and translated in vivo toproduce the antigen, which then stimulates an immune response in thesubject.

Prophylactic protection from hMPV/hPIV3 can be achieved followingadministration of the hMPV/hPIV3 mRNA vaccine of the present disclosure.Vaccines can be administered once, twice, three times, four times ormore but it is likely sufficient to administer the vaccine once(optionally followed by a single booster). It is possible, although lessdesirable, to administer the vaccine to an infected individual toachieve a therapeutic response. Dosing may need to be adjustedaccordingly.

A method of eliciting an immune response in a subject against hMPV/hPIV3is provided in aspects of the present disclosure. The method involvesadministering to the subject a hMPV/hPIV3 mRNA vaccine comprising atleast one RNA (e.g., mRNA) having an open reading frame encoding atleast one hMPV/hPIV3 antigen, thereby inducing in the subject an immuneresponse specific to a hMPV/hPIV3 antigen, wherein anti-antigen antibodytiter in the subject is increased following vaccination relative toanti-antigen antibody titer in a subject vaccinated with aprophylactically effective dose of a traditional vaccine against thehMPV/hPIV3. An “anti-antigen antibody” is a serum antibody the bindsspecifically to the antigen.

A prophylactically effective dose is an effective dose that preventsinfection with the virus at a clinically acceptable level. In someembodiments, the effective dose is a dose listed in a package insert forthe vaccine. A traditional vaccine, as used herein, refers to a vaccineother than the mRNA vaccines of the present disclosure. For instance, atraditional vaccine includes, but is not limited, to live microorganismvaccines, killed microorganism vaccines, subunit vaccines, proteinantigen vaccines, DNA vaccines, virus like particle (VLP) vaccines, etc.In exemplary embodiments, a traditional vaccine is a vaccine that hasachieved regulatory approval and/or is registered by a national drugregulatory body, for example the Food and Drug Administration (FDA) inthe United States or the European Medicines Agency (EMA).

In some embodiments, the anti-antigen antibody titer in the subject isincreased 1 log to 10 log following vaccination relative to anti-antigenantibody titer in a subject vaccinated with a prophylactically effectivedose of a traditional vaccine against the hMPV/hPIV3 or an unvaccinatedsubject. In some embodiments, the anti-antigen antibody titer in thesubject is increased 1 log, 2 log, 3 log, 4 log, 5 log, or 10 logfollowing vaccination relative to anti-antigen antibody titer in asubject vaccinated with a prophylactically effective dose of atraditional vaccine against the hMPV/hPIV3 or an unvaccinated subject.

A method of eliciting an immune response in a subject against hMPV/hPIV3is provided in other aspects of the disclosure. The method involvesadministering to the subject the hMPV/hPIV3 mRNA vaccine comprising atleast one RNA polynucleotide having an open reading frame encoding atleast one hMPV/hPIV3 antigen, thereby inducing in the subject an immuneresponse specific to hMPV/hPIV3 antigen, wherein the immune response inthe subject is equivalent to an immune response in a subject vaccinatedwith a traditional vaccine against the hMPV/hPIV3 at 2 times to 100times the dosage level relative to the RNA vaccine.

In some embodiments, the immune response in the subject is equivalent toan immune response in a subject vaccinated with a traditional vaccine attwice the dosage level relative to the hMPV/hPIV3 mRNA vaccine. In someembodiments, the immune response in the subject is equivalent to animmune response in a subject vaccinated with a traditional vaccine atthree times the dosage level relative to the hMPV/hPIV3 mRNA vaccine. Insome embodiments, the immune response in the subject is equivalent to animmune response in a subject vaccinated with a traditional vaccine at 4times, 5 times, 10 times, 50 times, or 100 times the dosage levelrelative to the hMPV/hPIV3 mRNA vaccine. In some embodiments, the immuneresponse in the subject is equivalent to an immune response in a subjectvaccinated with a traditional vaccine at 10 times to 1000 times thedosage level relative to the hMPV/hPIV3 mRNA vaccine. In someembodiments, the immune response in the subject is equivalent to animmune response in a subject vaccinated with a traditional vaccine at100 times to 1000 times the dosage level relative to the hMPV/hPIV3 mRNAvaccine.

In other embodiments, the immune response is assessed by determining[protein] antibody titer in the subject. In other embodiments, theability of serum or antibody from an immunized subject is tested for itsability to neutralize viral uptake or reduce hMPV/hPIV3 transformationof human B lymphocytes. In other embodiments, the ability to promote arobust T cell response(s) is measured using art recognized techniques.

Other aspects the disclosure provide methods of eliciting an immuneresponse in a subject against hMPV/hPIV3 by administering to the subjectthe hMPV/hPIV3 mRNA vaccine comprising at least one RNA polynucleotidehaving an open reading frame encoding at least one hMPV/hPIV3 antigen,thereby inducing in the subject an immune response specific tohMPV/hPIV3 antigen, wherein the immune response in the subject isinduced 2 days to 10 weeks earlier relative to an immune responseinduced in a subject vaccinated with a prophylactically effective doseof a traditional vaccine against hMPV/hPIV3. In some embodiments, theimmune response in the subject is induced in a subject vaccinated with aprophylactically effective dose of a traditional vaccine at 2 times to100 times the dosage level relative to the RNA vaccine.

In some embodiments, the immune response in the subject is induced 2days, 3 days, 1 week, 2 weeks, 3 weeks, 5 weeks, or 10 weeks earlierrelative to an immune response induced in a subject vaccinated with aprophylactically effective dose of a traditional vaccine.

Also provided herein are methods of eliciting an immune response in asubject against a hMPV/hPIV3 by administering to the subject thehMPV/hPIV3 mRNA vaccine having an open reading frame encoding a firstantigen, wherein the RNA polynucleotide does not include a stabilizationelement, and wherein an adjuvant is not co-formulated or co-administeredwith the vaccine.

The hMPV/hPIV3 mRNA vaccine may be administered by any route whichresults in a therapeutically effective outcome. These include, but arenot limited, to intradermal, intramuscular, intranasal, and/orsubcutaneous administration. The present disclosure provides methodscomprising administering RNA vaccines to a subject in need thereof. Theexact amount required will vary from subject to subject, depending onthe species, age, and general condition of the subject, the severity ofthe disease, the particular composition, its mode of administration, itsmode of activity, and the like. The hMPV/hPIV3 mRNA vaccine is typicallyformulated in dosage unit form for ease of administration and uniformityof dosage. It will be understood, however, that the total daily usage ofthe hMPV/hPIV3 mRNA vaccine may be decided by the attending physicianwithin the scope of sound medical judgment. The specific therapeuticallyeffective, prophylactically effective, or appropriate imaging dose levelfor any particular patient will depend upon a variety of factorsincluding the disorder being treated and the severity of the disorder;the activity of the specific compound employed; the specific compositionemployed; the age, body weight, general health, sex and diet of thepatient; the time of administration, route of administration, and rateof excretion of the specific compound employed; the duration of thetreatment; drugs used in combination or coincidental with the specificcompound employed; and like factors well known in the medical arts.

In some embodiments, the subject is an adult subject. An adult subjectis any human subject who has an age of 18 years or older. In someembodiments, an adult subject is between the ages of 18 to 49 years(e.g., 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or 49years). In some embodiments, an adult subject is between the ages of 18and 65 years. In some embodiments, an adult subject is a geriatricsubject who has an age of at least 65 years.

In other embodiments, the subject is a pediatric subject. A pediatricsubject is any human subject who has an age of younger than 18 years. Insome embodiments, a pediatric subject is between the ages of 12 monthsand 36 months. In some embodiments, a pediatric subject is between theages of 1 year and 17 years (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, or 17 years). In some embodiments, a pediatricsubject has an age of 10 years or younger (e.g., 6 months to 10 years,or 12 months to 10 years). In some embodiments, a pediatric subject hasan age of 5 years or younger (e.g., 6 months to 5 years, or 12 months to5 years).

The effective amount of the hMPV/hPIV3 mRNA vaccine, as provided herein,may be as low as 10 μg, administered for example as a single dose or astwo 5 μg doses. In some embodiments, the effective amount of thehMPV/hPIV3 mRNA vaccine, as provided herein, may be as low as 20 μg,administered for example as a single dose or as two 10 μg doses. In someembodiments, the effective amount is a total dose of 10 μg-300 μg or 20μg-300 μg or 25 μg-300 μg. For example, the effective amount may be atotal dose of 10 μg, 15 μg, 20 μg, 25 μg, 30 μg, 35 μg, 40 μg, 45 μg, 50μg, 55 μg, 60 μg, 65 μg, 70 μg, 75 μg, 80 μg, 85 μg, 90 μg, 95 μg, 100μg, 110 μg, 120 μg, 130 μg, 140 μg, 160 μg, 170 μg, 180 μg, 190 μg, 200μg, 250 μg, or 300 μg. In some embodiments, the effective amount is atotal dose of 10 μg-300 μg. In some embodiments, the effective amount isa total dose of 10 μg. In some embodiments, the effective amount is atotal dose of 20 μg. In some embodiments, the effective amount is atotal dose of 25 μg. In some embodiments, the effective amount is atotal dose of 30 μg. In some embodiments, the effective amount is atotal dose of 75 μg. In some embodiments, the effective amount is atotal dose of 100 μg. In some embodiments, the effective amount is atotal dose of 150 μg. In some embodiments, the effective amount is atotal dose of 300 μg.

In some embodiments, the hMPV/hPIV3 mRNA vaccine is administered to anadult human subject. The effective amount of the hMPV/hPIV3 mRNA vaccinefor the adult subject, as provided herein, may be as low as 20 μg,administered for example as a single dose or as two 10 μg doses. In someembodiments, the effective amount is a total dose of 20 μg-300 μg, 25μg-300 μg, or 30 μg-300 μg. For example, the effective amount may be atotal dose of 20 μg, 25 μg, 30 μg, 35 μg, 40 μg, 45 μg, 50 μg, 55 μg, 60μg, 65 μg, 70 μg, 75 μg, 80 μg, 85 μg, 90 μg, 95 μg, 100 μg, 110 μg, 120μg, 130 μg, 140 μg, 150 μg, 160 μg, 170 μg, 180 μg, 190 μg, 200 μg, 250μg, or 300 μg. In some embodiments, the effective amount is a total doseof 25 μg-300 μg.

In some embodiments, the effective amount is a total dose of 20 μg. Insome embodiments, the effective amount is a total dose of 30 μg. In someembodiments, the effective amount is a total dose of 25 μg. In someembodiments, the effective amount is a total dose of 75 μg. In someembodiments, the effective amount is a total dose of 150 μg. In someembodiments, the effective amount is a total dose of 300 μg.

In some embodiments, the hMPV/hPIV3 mRNA vaccine is administered topediatric human subject. The effective amount of the hMPV/hPIV3 mRNAvaccine for the pediatric subject, as provided herein, may be as low as10μg, administered for example as a single dose or as two 5μg doses. Insome embodiments, the effective amount is a total dose of 10 μg-150μg or20 μg-150μg or 30 μg-150 μg. For example, the effective amount may be atotal dose of 10 μg, 15 μg, 20 μg, 25 μg, 30 μg, 35 μg, 40 μg, 45 μg, 50μg, 55 μg, 60 μg, 65 μg, 70 μg, 75 μg, 80 μg, 85 μg, 90 μg, 95 μg, 100μg, 110 μg, 120 μg, 130 μg, 140 μg, or 150 μg. In some embodiments, theeffective amount is a total dose of 10 μg-150 μg. In some embodiments,the effective amount is a total dose of 10 μg. In some embodiments, theeffective amount is a total dose of 30 μg. In some embodiments, theeffective amount is a total dose of 100 μg.

The hMPV/hPIV3 mRNA vaccine described herein can be formulated into adosage form described herein, such as an intranasal, intratracheal, orinjectable (e.g., intravenous, intraocular, intravitreal, intramuscular,intradermal, intracardiac, intraperitoneal, and subcutaneous).

Vaccine Efficacy

Some aspects of the present disclosure provide formulations of thehMPV/hPIV3 mRNA vaccine, wherein the hMPV/hPIV3 mRNA vaccine isformulated in an effective amount to produce an antigen specific immuneresponse in a subject (e.g., production of antibodies specific to ananti-hMPV/hPIV3 antigen). “An effective amount” is a dose of thehMPV/hPIV3 mRNA vaccine effective to produce an antigen-specific immuneresponse. Also provided herein are methods of inducing anantigen-specific immune response in a subject.

As used herein, an immune response to a vaccine or LNP of the presentdisclosure is the development in a subject of a humoral and/or acellular immune response to a (one or more) hMPV/hPIV3 protein(s)present in the vaccine. For purposes of the present disclosure, a“humoral” immune response refers to an immune response mediated byantibody molecules, including, e.g., secretory (IgA) or IgG molecules,while a “cellular” immune response is one mediated by T-lymphocytes(e.g., CD4+ helper and/or CD8+ T cells (e.g., CTLs) and/or other whiteblood cells. One important aspect of cellular immunity involves anantigen-specific response by cytolytic T-cells (CTLs). CTLs havespecificity for peptide antigens that are presented in association withproteins encoded by the major histocompatibility complex (MHC) andexpressed on the surfaces of cells. CTLs help induce and promote thedestruction of intracellular microbes or the lysis of cells infectedwith such microbes. Another aspect of cellular immunity involves andantigen-specific response by helper T-cells. Helper T-cells act to helpstimulate the function, and focus the activity nonspecific effectorcells against cells displaying peptide antigens in association with MHCmolecules on their surface. A cellular immune response also leads to theproduction of cytokines, chemokines, and other such molecules producedby activated T-cells and/or other white blood cells including thosederived from CD4+ and CD8+ T-cells.

In some embodiments, the antigen-specific immune response ischaracterized by measuring an anti-hMPV/hPIV3 antigen antibody titerproduced in a subject administered the hMPV/hPIV3 mRNA vaccine asprovided herein. An antibody titer is a measurement of the amount ofantibodies within a subject, for example, antibodies that are specificto a particular antigen (e.g., an anti-hMPV/hPIV3 antigen) or epitope ofan antigen. Antibody titer is typically expressed as the inverse of thegreatest dilution that provides a positive result. Enzyme-linkedimmunosorbent assay (ELISA) is a common assay for determining antibodytiters, for example.

In some embodiments, an antibody titer is used to assess whether asubject has had an infection or to determine whether immunizations arerequired. In some embodiments, an antibody titer is used to determinethe strength of an autoimmune response, to determine whether a boosterimmunization is needed, to determine whether a previous vaccine waseffective, and to identify any recent or prior infections. In accordancewith the present disclosure, an antibody titer may be used to determinethe strength of an immune response induced in a subject by thehMPV/hPIV3 mRNA vaccine.

In some embodiments, an anti-hMPV/hPIV3 antigen antibody titer producedin a subject is increased by at least 1 log relative to a control. Forexample, anti-hMPV/hPIV3 antigen antibody titer produced in a subjectmay be increased by at least 1.5, at least 2, at least 2.5, or at least3 log relative to a control. In some embodiments, the anti-hMPV/hPIV3antigen antibody titer produced in the subject is increased by 1, 1.5,2, 2.5 or 3 log relative to a control. In some embodiments, theanti-hMPV/hPIV3 antigen antibody titer produced in the subject isincreased by 1-3 log relative to a control. For example, theanti-hMPV/hPIV3 antigen antibody titer produced in a subject may beincreased by 1-1.5, 1-2, 1-2.5, 1-3, 1.5-2, 1.5-2.5, 1.5-3, 2-2.5, 2-3,or 2.5-3 log relative to a control.

In some embodiments, the anti-hMPV/hPIV3 antigen antibody titer producedin a subject is increased at least 2 times relative to a control. Forexample, the anti-hMPV/hPIV3 antigen antibody titer produced in asubject may be increased at least 3 times, at least 4 times, at least 5times, at least 6 times, at least 7 times, at least 8 times, at least 9times, or at least 10 times relative to a control. In some embodiments,the anti-hMPV/hPIV3 antigen antibody titer produced in the subject isincreased 2, 3, 4, 5, 6, 7, 8, 9, or 10 times relative to a control. Insome embodiments, the anti-hMPV/hPIV3 antigen antibody titer produced ina subject is increased 2-10 times relative to a control. For example,the anti-hMPV/hPIV3 antigen antibody titer produced in a subject may beincreased 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-10, 3-9, 3-8, 3-7,3-6, 3-5, 3-4, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, 5-6,6-10, 6-9, 6-8, 6-7, 7-10, 7-9, 7-8, 8-10, 8-9, or 9-10 times relativeto a control.

In some embodiments, an antigen-specific immune response is measured asa ratio of geometric mean titer (GMT), referred to as a geometric meanratio (GMR), of serum neutralizing antibody titers to hMPV and hPIV3. Ageometric mean titer (GMT) is the average antibody titer for a group ofsubjects calculated by multiplying all values and taking the nth root ofthe number, where n is the number of subjects with available data.

In some embodiments, the GMR of 28 days to baseline titers for hMPV(e.g., hMPV-A and/or hMPV-B) in subjects administered a ≥25 μg, ≥30 μg,≥75 μg, ≥150 μg, or ≥300 μg dose of the vaccine composition is in therange of 4 to 8. In some embodiments, the GMR of 28 days to baselinetiters for hMPV (e.g., hMPV-A and/or hMPV-B) in pediatric subjectsadministered a ≥10 μg, ≥30 μg, or ≥100 μg dose of the vaccinecomposition is in the range of 3 to 9. In some embodiments, the GMR of28 days to baseline titers for hMPV (e.g., hMPV-A and/or hMPV-B) insubjects administered a ≥75 μg dose of the vaccine composition is in therange of 4 to 8. For example, the GMR of 28 days to baseline titers forhMPV in subjects administered a ≥25 μg, ≥30 μg, ≥75 μg, ≥150 μg, or ≥300μg dose of the vaccine composition may be 3, 3.5, 4, 4.5, 5, 5.5, 6,6.5, 7, 7.5, 8, 8.5, or 9. In some embodiments, the GMR of 28 days tobaseline titers for hMPV in pediatric subjects administered a ≥10 μg,≥30 μg, or ≥100 μg dose of the vaccine composition may be 3, 3.5, 4,4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, or 9. In some embodiments, the GMRof 28 days to baseline titers for hMPV in subjects administered a ≥25μg, ≥30 μg, ≥75 μg, ≥150 μg, or ≥300 μg dose of the vaccine compositionis 3.53-8.52. In some embodiments, the GMR of 28 days to baseline titersfor hMPV in subjects administered a ≥25 μg, ≥30 μg, ≥75 μg, ≥150 μg, or≥300 μg dose of the vaccine composition is 6-6.5. For example, the GMRof 28 days to baseline titers for hMPV in subjects administered a ≥25μg, ≥30 μg, ≥75 μg, ≥150 μg, or ≥300 μg dose of the vaccine compositionmay be 6, 6.1, 6.15, 6.2, 6.25, 6.3, 6.35, 6.4, 6.45, or 6.5. In someembodiments, the GMR of 28 days to baseline titers for hMPV (e.g.,hMPV-A) in subjects administered a ≥25 μg, ≥30 μg, ≥75 μg, ≥150 μg, or≥300 μg dose of the vaccine composition is 6.04. In some embodiments,the GMR of 28 days to baseline titers for hMPV (e.g., hMPV-B) insubjects administered a ≥25 μg, ≥30 μg, ≥75 μg, ≥150 μg, or ≥300 μg doseof the vaccine composition is 6.33.

In some embodiments, the GMR of 28 days to baseline titers for hPIV3 insubjects administered a ≥25 μg, ≥30 μg, ≥75 μg, ≥150 μg, or ≥300 μg doseof the vaccine composition is in the range of 2 to 5. In someembodiments, the GMR of 28 days to baseline titers for hPIV3 inpediatric subjects administered a ≥10 μg, ≥30 μg, or ≥100 μg dose of thevaccine composition is in the range of 2 to 5. In some embodiments, theGMR of 28 days to baseline titers for hPIV3 in subjects administered a≥75 μg dose of the vaccine composition is in the range of 2 to 5. Forexample, the GMR of 28 days to baseline titers for hPIV3 in subjectsadministered a ≥25 μg, ≥30 μg, ≥75 μg, ≥150 μg, or ≥300 μg dose of thevaccine composition may be 2, 2.5, 3, 3.5, 4, 4.5, or 5. In someembodiments, the GMR of 28 days to baseline titers for hPIV3 inpediatric subjects administered a ≥10 μg, ≥30 μg, or ≥100 μg dose of thevaccine composition may be 2, 2.5, 3, 3.5, 4, 4.5, or 5. In someembodiments, the GMR of 28 days to baseline titers for hPIV3 in subjectsadministered a ≥25 μg, ≥30 μg, ≥75 μg, ≥150 μg, or ≥300 μg dose of thevaccine composition is 2.67-3.36. For example, the GMR of 28 days tobaseline titers for hPIV3 in subjects administered a ≥25 μg, ≥30 μg, ≥75μg, ≥150 μg, or ≥300 μg dose of the vaccine composition may be 2.6,2.65, 2.7, 2.75, 2.8, 2.85, 2.9, 2.95, 3, 3.05, 3.1, 3.15, 3.2, 3.25,3.3, 3.35, 3.4, 3.45, 3.5, or 3.55. In some embodiments, the GMR of 28days to baseline titers for hPIV3 in subjects administered a ≥25 μg, ≥30μg, ≥75 μg, ≥150 μg, or ≥300 μg dose of the vaccine composition is 3.24.

In some embodiments, the GMR of 28 days to baseline titers for hMPV-A insubjects administered a 75 μg dose of the vaccine composition is in therange of 4 to 6, e.g., 5.07. In some embodiments, the GMR of 28 days tobaseline titers for hMPV-A in subjects administered a 150 μg dose of thevaccine composition is in the range of 4.5 to 6.5, e.g., 5.84. In someembodiments, the GMR of 28 days to baseline titers for hMPV-A insubjects administered a 300 μg dose of the vaccine composition is in therange of 6 to 8, e.g., 7.09. In some embodiments, the GMR of 56 days tobaseline titers for hMPV-A in subjects administered a 75 μg dose of thevaccine composition is in the range of 3 to 5, e.g., 3.87. In someembodiments, the GMR of 56 days to baseline titers for hMPV-A insubjects administered a 150 μg dose of the vaccine composition is in therange of 3 to 5, e.g., 3.18. In some embodiments, the GMR of 56 days tobaseline titers for hMPV-A in subjects administered a 300 μg dose of thevaccine composition is in the range of 5.5 to 7.5, e.g., 6.05. In someembodiments, the GMR of 196 days to baseline titers for hMPV-A insubjects administered a 75 μg dose of the vaccine composition is in therange of 1 to 3, e.g., 1.82. In some embodiments, the GMR of 196 days tobaseline titers for hMPV-A in subjects administered a 150 μg dose of thevaccine composition is in the range of 2 to 4, e.g., 2.08. In someembodiments, the GMR of 196 days to baseline titers for hMPV-A insubjects administered a 300 μg dose of the vaccine composition is in therange of 2.5 to 4.5, e.g., 3.33.

In some embodiments, the GMR of 28 days to baseline titers for hMPV-B insubjects administered a 75 μg dose of the vaccine composition is in therange of 3.5 to 5.5, e.g., 4.87. In some embodiments, the GMR of 28 daysto baseline titers for hMPV-B in subjects administered a 150 μg dose ofthe vaccine composition is in the range of 6.5 to 8.5, e.g., 7.73. Insome embodiments, the GMR of 28 days to baseline titers for hMPV-B insubjects administered a 300 μg dose of the vaccine composition is in therange of 6 to 8, e.g., 7.01. In some embodiments, the GMR of 56 days tobaseline titers for hMPV-B in subjects administered a 75 μg dose of thevaccine composition is in the range of 3 to 5, e.g., 4.14. In someembodiments, the GMR of 56 days to baseline titers for hMPV-B insubjects administered a 150 μg dose of the vaccine composition is in therange of 6 to 8, e.g., 6.58. In some embodiments, the GMR of 56 days tobaseline titers for hMPV-B in subjects administered a 300 μg dose of thevaccine composition is in the range of 3.5 to 5.5, e.g., 4.24. In someembodiments, the GMR of 196 days to baseline titers for hMPV-B insubjects administered a 75 μg dose of the vaccine composition is in therange of 2 to 5, e.g., 3.06. In some embodiments, the GMR of 196 days tobaseline titers for hMPV-B in subjects administered a 150 μg dose of thevaccine composition is in the range of 3 to 5, e.g., 3.47. In someembodiments, the GMR of 196 days to baseline titers for hMPV-B insubjects administered a 300 μg dose of the vaccine composition is in therange of 3 to 5, e.g., 3.93.

In some embodiments, the GMR of 28 days to baseline titers for hPIV3 insubjects administered a 75 μg dose of the vaccine composition is in therange of 2.5 to 4.5, e.g., 3.36. In some embodiments, the GMR of 28 daysto baseline titers for hPIV3 in subjects administered a 150 μg dose ofthe vaccine composition is in the range of 2 to 4, e.g., 3.13. In someembodiments, the GMR of 28 days to baseline titers for hPIV3 in subjectsadministered a 300 μg dose of the vaccine composition is in the range of2 to 4, e.g., 3.29. In some embodiments, the GMR of 56 days to baselinetiters for hPIV3 in subjects administered a 75 μg dose of the vaccinecomposition is in the range of 2.5 to 4.5, e.g., 3.34. In someembodiments, the GMR of 56 days to baseline titers for hPIV3 in subjectsadministered a 150 μg dose of the vaccine composition is in the range of1 to 3, e.g., 1.97. In some embodiments, the GMR of 56 days to baselinetiters for hPIV3 in subjects administered a 300 μg dose of the vaccinecomposition is in the range of 2.5 to 4.5, e.g., 3.23. In someembodiments, the GMR of 196 days to baseline titers for hPIV3 insubjects administered a 75 μg dose of the vaccine composition is in therange of 1 to 3, e.g., 1.76. In some embodiments, the GMR of 196 days tobaseline titers for hPIV3 in subjects administered a 150 μg dose of thevaccine composition is in the range of 1 to 3, e.g., 1.26. In someembodiments, the GMR of 196 days to baseline titers for hPIV3 insubjects administered a 300 μg dose of the vaccine composition is in therange of 1 to 3, e.g., 1.73.

In some embodiments, the GMR of 28 days to baseline titers for hMPV-A insubjects administered two 75 μg doses of the vaccine composition is inthe range of 6 to 8, e.g., 7.06. In some embodiments, the GMR of 28 daysto baseline titers for hMPV-A in subjects administered two 150 μg dosesof the vaccine composition is in the range of 7 to 9, e.g., 8.16. Insome embodiments, the GMR of 28 days to baseline titers for hMPV-A insubjects administered two 300 μg doses of the vaccine composition is inthe range of 6 to 8, e.g., 6.99. In some embodiments, the GMR of 56 daysto baseline titers for hMPV-A in subjects administered two 75 μg dosesof the vaccine composition is in the range of 6.5 to 8.5, e.g., 7.56. Insome embodiments, the GMR of 56 days to baseline titers for hMPV-A insubjects administered two 150 μg doses of the vaccine composition is inthe range of 8.5 to 10.5, e.g., 9.82. In some embodiments, the GMR of 56days to baseline titers for hMPV-A in subjects administered two 300 μgdoses of the vaccine composition is in the range of 5.5 to 7.5, e.g.,5.86. In some embodiments, the GMR of 196 days to baseline titers forhMPV-A in subjects administered two 75 μg doses of the vaccinecomposition is in the range of 2 to 4, e.g., 3.10. In some embodiments,the GMR of 196 days to baseline titers for hMPV-A in subjectsadministered two 150 μg doses of the vaccine composition is in the rangeof 3.5 to 5.5, e.g., 4.48. In some embodiments, the GMR of 196 days tobaseline titers for hMPV-A in subjects administered two 300 μg doses ofthe vaccine composition is in the range of 2 to 4, e.g., 2.89.

In some embodiments, the GMR of 28 days to baseline titers for hMPV-B insubjects administered two 75 μg doses of the vaccine composition is inthe range of 3.5 to 5.5, e.g., 4.38. In some embodiments, the GMR of 28days to baseline titers for hMPV-B in subjects administered two 150 μgdoses of the vaccine composition is in the range of 6.5 to 8.5, e.g.,7.80. In some embodiments, the GMR of 28 days to baseline titers forhMPV-B in subjects administered two 300 μg doses of the vaccinecomposition is in the range of 7.5 to 9.5, e.g., 8.65. In someembodiments, the GMR of 56 days to baseline titers for hMPV-B insubjects administered two 75 μg doses of the vaccine composition is inthe range of 4 to 6, e.g., 5.06. In some embodiments, the GMR of 56 daysto baseline titers for hMPV-B in subjects administered two 150 μg dosesof the vaccine composition is in the range of 9.5 to 11.5, e.g., 10.59.In some embodiments, the GMR of 56 days to baseline titers for hMPV-B insubjects administered two 300 μg doses of the vaccine composition is inthe range of 7 to 9, e.g., 8.30. In some embodiments, the GMR of 196days to baseline titers for hMPV-B in subjects administered two 75 μgdoses of the vaccine composition is in the range of 1 to 3, e.g., 2.08.In some embodiments, the GMR of 196 days to baseline titers for hMPV-Bin subjects administered two 150 μg doses of the vaccine composition isin the range of 5 to 7, e.g., 6.22. In some embodiments, the GMR of 196days to baseline titers for hMPV-B in subjects administered two 300 μgdoses of the vaccine composition is in the range of 3 to 5, e.g., 4.23.

In some embodiments, the GMR of 28 days to baseline titers for hPIV3 insubjects administered two 75 μg doses of the vaccine composition is inthe range of 2 to 4, e.g., 2.78. In some embodiments, the GMR of 28 daysto baseline titers for hPIV3 in subjects administered two 150 μg dosesof the vaccine composition is in the range of 2 to 4, e.g., 3.39. Insome embodiments, the GMR of 28 days to baseline titers for hPIV3 insubjects administered two 300 μg doses of the vaccine composition is inthe range of 2.5 to 4.5, e.g., 3.54. In some embodiments, the GMR of 56days to baseline titers for hPIV3 in subjects administered two 75 μgdoses of the vaccine composition is in the range of 2.5 to 4.5, e.g.,3.60. In some embodiments, the GMR of 56 days to baseline titers forhPIV3 in subjects administered two 150 μg doses of the vaccinecomposition is in the range of 2.5 to 4.5, e.g., 3.74. In someembodiments, the GMR of 56 days to baseline titers for hPIV3 in subjectsadministered two 300 μg doses of the vaccine composition is in the rangeof 3.5 to 5.5, e.g., 4.76. In some embodiments, the GMR of 196 days tobaseline titers for hPIV3 in subjects administered two 75 μg doses ofthe vaccine composition is in the range of 1 to 3, e.g., 1.87. In someembodiments, the GMR of 196 days to baseline titers for hPIV3 insubjects administered two 150 μg doses of the vaccine composition is inthe range of 1 to 3, e.g., 2.33. In some embodiments, the GMR of 196days to baseline titers for hPIV3 in subjects administered two 300 μgdoses of the vaccine composition is in the range of 2.5 to 4.5, e.g.,3.52.

In some embodiments, the geometric mean titer (GMT) of serumneutralizing antibodies to hMPV increases in the subject by at least2-fold within 30 days relative to baseline. For example, the GMT ofserum neutralizing antibodies to hMPV may increase in the subject by atleast 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, atleast 7-fold, at least 8-fold, at least 9-fold, or at least 10-foldwithin 30 days relative to baseline. In some embodiments, the GMT ofserum neutralizing antibodies to hMPV increases in the subject by 2-foldto 10-fold within 30 days relative to baseline. In some embodiments, theincrease in GMT of serum neutralizing antibodies to hMPV follows asingle 25 μg dose of the vaccine composition. In other embodiments, theincrease in GMT of serum neutralizing antibodies to hMPV follows asingle 50 μg dose of the vaccine composition. In yet other embodiments,the increase in GMT of serum neutralizing antibodies to hMPV follows asingle 75 μg dose of the vaccine composition. For example, the GMT inserum neutralizing antibodies to hMPV may increase in the subject by atleast 2-fold within 30 days relative to baseline, following a single 25μg dose of the vaccine composition. As another example, the GMT in serumneutralizing antibodies to hMPV may increase in the subject by at least2-fold within 30 days relative to baseline, following a single 75 μgdose. As yet another example, the GMT in serum neutralizing antibodiesto hMPV may increase in the subject by at least 6-fold within 30 daysrelative to baseline, following a single 25 μg dose of the vaccinecomposition. As still another example, the GMT in serum neutralizingantibodies to hMPV may increase in the subject by at least 6-fold within30 days relative to baseline, following a single 75 μg dose.

In some embodiments, the geometric mean titer (GMT) of serumneutralizing antibodies to hPIV3 increases in the subject by at least2-fold within 30 days relative to baseline. For example, the GMT ofserum neutralizing antibodies to hPIV3 may increase in the subject by atleast 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, atleast 7-fold, at least 8-fold, at least 9-fold, or at least 10-foldwithin 30 days relative to baseline. In some embodiments, the GMT ofserum neutralizing antibodies to hPIV3 increases in the subject by2-fold to 10-fold within 30 days relative to baseline. In someembodiments, the increase in GMT of serum neutralizing antibodies tohPIV3 follows a single 25 μg dose of the vaccine composition. In otherembodiments, the increase in GMT of serum neutralizing antibodies tohPIV3 follows a single 50 μg dose of the vaccine composition. In yetother embodiments, the increase in GMT of serum neutralizing antibodiesto hPIV3 follows a single 75 μg dose of the vaccine composition. Forexample, the GMT in serum neutralizing antibodies to hPIV3 may increasein the subject by at least 2-fold within 30 days relative to baseline,following a single 25 μg dose of the vaccine composition. As anotherexample, the GMT in serum neutralizing antibodies to hPIV3 may increasein the subject by at least 2-fold within 30 days relative to baseline,following a single 75 μg dose. As yet another example, the GMT in serumneutralizing antibodies to hPIV3 may increase in the subject by at least3-fold within 30 days relative to baseline, following a single 25 μgdose of the vaccine composition. As still another example, the GMT inserum neutralizing antibodies to hPIV3 may increase in the subject by atleast 3-fold within 30 days relative to baseline, following a single 75μg dose.

A control, in some embodiments, is the anti-hMPV/hPIV3 antigen antibodytiter produced in a subject who has not been administered the hMPV/hPIV3mRNA vaccine. In some embodiments, a control is an anti-hMPV/hPIV3antigen antibody titer produced in a subject administered a recombinantor purified hMPV/hPIV3protein vaccine. Recombinant protein vaccinestypically include protein antigens that either have been produced in aheterologous expression system (e.g., bacteria or yeast) or purifiedfrom large amounts of the pathogenic organism.

In some embodiments, the ability of the hMPV/hPIV3 mRNA vaccine to beeffective is measured in a murine model. For example, the hMPV/hPIV3mRNA vaccine may be administered to a murine model and the murine modelassayed for induction of neutralizing antibody titers. Viral challengestudies may also be used to assess the efficacy of a vaccine of thepresent disclosure. For example, the hMPV/hPIV3 mRNA vaccine may beadministered to a murine model, the murine model challenged withhMPV/hPIV3, and the murine model assayed for survival and/or immuneresponse (e.g., neutralizing antibody response, T cell response (e.g.,cytokine response)).

In some embodiments, an effective amount of the hMPV/hPIV3 mRNA vaccineis a dose that is reduced compared to the standard of care dose of arecombinant hMPV/hPIV3protein vaccine. A “standard of care,” as providedherein, refers to a medical or psychological treatment guideline and canbe general or specific. “Standard of care” specifies appropriatetreatment based on scientific evidence and collaboration between medicalprofessionals involved in the treatment of a given condition. It is thediagnostic and treatment process that a physician/clinician shouldfollow for a certain type of patient, illness or clinical circumstance.A “standard of care dose,” as provided herein, refers to the dose of arecombinant or purified hMPV/hPIV3protein vaccine, or a live attenuatedor inactivated hMPV/hPIV3vaccine, or a hMPV/hPIV3VLP vaccine, that aphysician/clinician or other medical professional would administer to asubject to treat or prevent hMPV/hPIV3, or a hMPV/hPIV3-relatedcondition, while following the standard of care guideline for treatingor preventing hMPV/hPIV3, or a hMPV/hPIV3-related condition.

In some embodiments, the anti-hMPV/hPIV3 antigen antibody titer producedin a subject administered an effective amount of the hMPV/hPIV3 mRNAvaccine is equivalent to an anti-hMPV/hPIV3 antigen antibody titerproduced in a control subject administered a standard of care dose of arecombinant or purified hMPV/hPIV3 protein vaccine, or a live attenuatedor inactivated hMPV/hPIV3 vaccine, or a hMPV/hPIV3 VLP vaccine.

Vaccine efficacy may be assessed using standard analyses (see, e.g.,Weinberg et al., J Infect Dis. 2010 Jun. 1; 201 (11):1607-10). Forexample, vaccine efficacy may be measured by double-blind, randomized,clinical controlled trials. Vaccine efficacy may be expressed as aproportionate reduction in disease attack rate (AR) between theunvaccinated (ARU) and vaccinated (ARV) study cohorts and can becalculated from the relative risk (RR) of disease among the vaccinatedgroup with use of the following formulas:

Efficacy=(ARU−ARV)/ARU×100; and

Efficacy=(1−RR)×100.

Likewise, vaccine effectiveness may be assessed using standard analyses(see, e.g., Weinberg et al., J Infect Dis. 2010 Jun. 1; 201(11):1607-10). Vaccine effectiveness is an assessment of how a vaccine(which may have already proven to have high vaccine efficacy) reducesdisease in a population. This measure can assess the net balance ofbenefits and adverse effects of a vaccination program, not just thevaccine itself, under natural field conditions rather than in acontrolled clinical trial. Vaccine effectiveness is proportional tovaccine efficacy (potency) but is also affected by how well targetgroups in the population are immunized, as well as by othernon-vaccine-related factors that influence the ‘real-world’ outcomes ofhospitalizations, ambulatory visits, or costs. For example, aretrospective case control analysis may be used, in which the rates ofvaccination among a set of infected cases and appropriate controls arecompared. Vaccine effectiveness may be expressed as a rate difference,with use of the odds ratio (OR) for developing infection despitevaccination:

Effectiveness=(1−OR)×100.

In some embodiments, efficacy of the hMPV/hPIV3 mRNA vaccine is at least60% relative to unvaccinated control subjects. For example, efficacy ofthe hMPV/hPIV3 mRNA vaccine may be at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 95%, at least 98%, or 100%relative to unvaccinated control subjects.

Sterilizing Immunity. Sterilizing immunity refers to a unique immunestatus that prevents effective pathogen infection into the host. In someembodiments, the effective amount of a the hMPV/hPIV3 mRNA vaccine ofthe present disclosure is sufficient to provide sterilizing immunity inthe subject for at least 1 year. For example, the effective amount ofthe hMPV/hPIV3 mRNA vaccine of the present disclosure is sufficient toprovide sterilizing immunity in the subject for at least 2 years, atleast 3 years, at least 4 years, or at least 5 years. In someembodiments, the effective amount of the hMPV/hPIV3 mRNA vaccine of thepresent disclosure is sufficient to provide sterilizing immunity in thesubject at an at least 5-fold lower dose relative to control. Forexample, the effective amount may be sufficient to provide sterilizingimmunity in the subject at an at least 10-fold lower, 15-fold, or20-fold lower dose relative to a control.

Detectable Antigen. In some embodiments, the effective amount of thehMPV/hPIV3 mRNA vaccine of the present disclosure is sufficient toproduce detectable levels of hMPV/hPIV3 antigen as measured in serum ofthe subject at 1-72 hours post administration.

Titer. An antibody titer is a measurement of the amount of antibodieswithin a subject, for example, antibodies that are specific to aparticular antigen (e.g., an anti-hMPV/hPIV3 antigen). Antibody titer istypically expressed as the inverse of the greatest dilution thatprovides a positive result. Enzyme-linked immunosorbent assay (ELISA) isa common assay for determining antibody titers, for example.

In some embodiments, the effective amount of the hMPV/hPIV3 mRNA vaccineof the present disclosure is sufficient to produce a 1,000-10,000neutralizing antibody titer produced by neutralizing antibody againstthe hMPV/hPIV3 antigen as measured in serum of the subject at 1-72 hourspost administration. In some embodiments, the effective amount issufficient to produce a 1,000-5,000 neutralizing antibody titer producedby neutralizing antibody against the hMPV/hPIV3 antigen as measured inserum of the subject at 1-72 hours post administration. In someembodiments, the effective amount is sufficient to produce a5,000-10,000 neutralizing antibody titer produced by neutralizingantibody against the hMPV/hPIV3 antigen as measured in serum of thesubject at 1-72 hours post administration.

In some embodiments, the neutralizing antibody titer is at least 100NT50. For example, the neutralizing antibody titer may be at least 200,300, 400, 500, 600, 700, 800, 900 or 1000 NT₅₀. In some embodiments, theneutralizing antibody titer is at least 10,000 NT50.

In some embodiments, the neutralizing antibody titer is at least 100neutralizing units per milliliter (NU/mL). For example, the neutralizingantibody titer may be at least 200, 300, 400, 500, 600, 700, 800, 900 or1000 NU/mL. In some embodiments, the neutralizing antibody titer is atleast 10,000 NU/mL.

In some embodiments, an anti-hMPV/hPIV3 antigen antibody titer producedin the subject is increased by at least 1 log relative to a control. Forexample, an anti-hMPV/hPIV3 antigen antibody titer produced in thesubject may be increased by at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 logrelative to a control.

In some embodiments, an anti-hMPV/hPIV3 antigen antibody titer producedin the subject is increased at least 2 times relative to a control. Forexample, an anti-hMPV/hPIV3 antigen antibody titer produced in thesubject is increased by at least 3, 4, 5, 6, 7, 8, 9 or 10 timesrelative to a control.

In some embodiments, a geometric mean, which is the nth root of theproduct of n numbers, is generally used to describe proportional growth.Geometric mean, in some embodiments, is used to characterize antibodytiter produced in a subject.

A control may be, for example, an unvaccinated subject, or a subjectadministered a live attenuated hMPV/hPIV3 vaccine, an inactivatedhMPV/hPIV3 vaccine, or a protein subunit hMPV/hPIV3 vaccine.

EXAMPLES

Example 1. A Phase 1, Randomized, Observer-Blind, Placebo-Controlled,Dose-Ranging Study to Evaluate the Safety, Reactogenicity, andImmunogenicity of the mRNA hMPV/hPIV3 Vaccine, a Combined HumanMetapneumovirus (hMPV) and Human Parainfluenza Virus Type 3 (hPIV3)Vaccine, when Administered to Healthy Adults

This was a Phase 1, first-in-human (FIH), randomized, observer-blind,placebo-controlled, dose-ranging study to evaluate the safety,reactogenicity, and immunogenicity of a the combined hMPV and hPIV3 mRNAvaccine provided herein, administered intramuscularly (IM) according toa 1-dose versus 2-dose schedule in healthy adults (18 through 49 yearsof age).

Study Overview Study Design

The study was conducted in 2 phases, the dose-escalation phase and thedose-selection phase. All subjects are followed up for 1 year after thelast vaccination.

In the dose-escalation phase (FIGS. 1 and 2 ), there was sequentialenrollment of 5 subjects at each dose level (N=20), randomized in a 4:1ratio to receive either the hMPV/hPIV3 mRNA vaccine or placebo. Doselevels were 25, 75, 150, and 300 μg. Subjects were administered twodoses, at Day 1 and Month 1 (Day 28). Following Safety MonitoringCommittee (SMC) review of all safety and reactogenicity data up to Day35 of the dose-escalation phase, the 3 highest dose levels (75, 150, and300 μg) were selected to be evaluated in the dose-selection phase of thestudy.

In the dose-selection phase (FIGS. 1 and 3 ), subjects were randomlyassigned into one of 4 dose groups (75 μg hMPV/hPIV3 mRNA vaccine, 150μg hMPV/hPIV3 mRNA vaccine, 300 μg hMPV/hPIV3 mRNA vaccine, or placebo)in a 1:1:1:1 ratio, each cohort consisting of 26 subjects. Within eachhMPV/hPIV3 mRNA vaccine dose level group, subjects were randomlyassigned in a 1:1 ratio to receive the second dose of hMPV/hPIV3 mRNAvaccine or placebo at Month 1 (Day 28).

Objectives and Endpoints Primary Objectives

1. To evaluate the safety and reactogenicity of the hMPV/hPIV3 mRNAvaccine, administered according to a 1-dose versus 2-dose schedule,through 28 days after the last vaccination.

2. To evaluate the humoral immunogenicity of the hMPV/hPIV3 mRNAvaccine, administered according to a 1-dose versus 2-dose schedule,through 28 days after the last vaccination.

3. To select the optimal dose and vaccination schedule of the hMPV/hPIV3mRNA vaccine for further clinical development.

Primary Safety Endpoints

1. Occurrence of each solicited local and systemic adverse events (AE),during a 7-day follow-up period after each and any vaccination (i.e.,the day of vaccination and 6 subsequent days).

2. Occurrence of any unsolicited adverse events (AEs), serious adverseevents (SAEs), and adverse event of special interest (AESIs) that areconsidered related to the hMPV/hPIV3 mRNA vaccine during the entirestudy period (Day 1 through Month 13).

3. Occurrence of any laboratory abnormality at Day 1, Day 7, Month 1,Day 35, and Month 2.

4. Occurrence of any unsolicited AE, during a 28-day follow-up periodafter each study vaccination (the day of vaccination and 27 subsequentdays).

5. Occurrence of any medically-attended AEs from Day 1 to Month 2.

6. Occurrence of any AESIs from Day 1 to Month 2.

7. Occurrence of any SAEs from Day 1 to Month 2.

Primary Immunogenicity Endpoints

1. Geometric mean titer (GMT) of the serum anti-hMPV and anti-PIV3neutralizing antibodies at Day 1 (baseline), Month 1, and Month 2.

2. Proportion of subjects with a ≥4-fold increase in serum anti-hMPV andanti-PIV3 neutralizing antibody titer from Day 1 to Month 1 and Month 2.

3. Proportion of subjects at Month 1 and Month 2 who achieve serumanti-hMPV and anti-PIV3 neutralizing antibody titers greater than thethird quartile of the serum anti-hMPV and anti-PIV3 antibody titersoverall distribution at Day 1.

4. Reverse cumulative distribution of serum anti-hMPV and anti-PIV3neutralizing antibody titers at Day 1, Month 1, and Month 2.

Analyses

1. A 2-month interim analysis of safety, reactogenicity, andimmunogenicity data collected from Visit Day 1 to Visit Month 2 andassociated endpoints was conducted on cleaned data and reported on atreatment group level. This was a partially unblinded analysis in thataccess to individual listings was restricted to pre-identified Sponsorstudy team members. Study sites remained blinded.

2. A 7-month interim analysis of immunogenicity data collected fromVisit Day 1 to Visit Month 7 was conducted on cleaned data and wasreported on a treatment group level. This was a partially unblindedanalysis in that access to individual listings was restricted topre-identified Sponsor study team members. Study sites remained blinded.This analysis provided information about short-term antibodypersistence.

3. The final analysis of safety and immunogenicity data collected fromVisit Day 1 through the end of the study was performed as soon as thestudy database is cleaned and locked.

Results Demographics

Demographic and baseline characteristics were generally balanced acrosstreatment groups (Table 1). There were more females than males enrolledacross the treatment groups, except for the 300 μg 2-dose group, andbody mass index was higher in this group. Age was somewhat lower in the25 μg group, and there were more subjects of white race in the 75 μg2-dose and 300 μg 1-dose groups.

TABLE 1 Study Demographics hMPV/hPIV3 mRNA vaccine 25 μg 75 μg 150 μg300 μg Placebo 2-Dose 1-Dose 2-Dose 1-Dose 2-Dose 1-Dose 2-Dose TotalTotal (N = 30) (N = 4) (N = 13) (N = 17) (N = 13) (N = 17) (N = 13) (N =17) (N = 94) (N = 124) Age (years) Mean (SD) 39.7 28.0 36.4 39.6 34.335.6 34.9 35.1 35.7 36.7 (7.67) (9.38) (6.21) (6.85) (9.10) (7.98)(8.34) (9.47) (8.23) (8.25) Min, Max 19, 48 20, 38 25, 45 29, 49 20, 4722, 48 19, 47 19, 49 19, 49 19, 49 Gender, n (%) Male 12 (40.0) 1 (25.0)3 (23.1) 4 (23.5) 4 (30.8) 7 (41.2) 4 (30.8) 11 (64.7) 34 (36.2) 46(37.1) Female 18 (60.0) 3 (75.0) 10 (76.9) 13 (76.5) 9 (69.2) 10 (58.8)9 (69.2) 6 (35.3) 60 (63.8) 78 (62.9) Race, n (%) Am. Indian 1 (3.3) 0 00 0 0 0 0 0 1 (0.8) or Alaskan Native Asian 1 (3.3) 0 0 0 0 0 0 0 0 1(0.8) Black or 8 (26.7) 2 (50.0) 6 (46.2) 3 (17.6) 5 (38.5) 6 (35.3) 2(15.4) 8 (47.1) 32 (34.0) 40 (32.3) African American White 20 (66.7) 2(50.0) 7 (53.8) 14 (82.4) 8(61.5) 10 (58.8) 11 (84.6) 9 (52.9) 61 (64.9)81 (65.3) Multi- 0 0 0 0 0 1 (5.9) 0 0 1 (1.1) 1 (0.8) racial Ethnicity,n (%) Hispanic, 3 (10.0) 0 3 (23.1) 1 (5.9) 2 (15.4) 1 (5.9) 2 (15.4) 2(11.8) 11 (11.7) 14 (11.3) Latino, or Spanish Not 27 4 10 16 11 16 11 1583 110 Hispanic, (90.0) (100.0) (76.9) (94.1) (84.6) (94.1) (84.6)(88.2) (88.3) (88.7) Latino, or Spanish Weight (kg) Mean (SD) 77.2580.60 74.99 76.67 74.49 78.98 73.55 86.17 78.01 77.83 (13.885) (13.561)(10.651) (12.703) (15.598) (10.404) (11.881) (16.160) (13.425) (13.485)Min, Max 52.3, 60.3, 61.6, 51.2, 52.5, 63.2, 53.7, 51.7, 51.2, 51.2,114.0 88.5 103.2 95.5 101.2 94.9 91.1 119.0 119.0 119.0 Height (cm) Mean(SD) 168.81 175.58 167.35 166.45 168.72 167.88 165.29 173.61 168.67168.70 (8.502) (8.310) (8.055) (9.071) (8.342) (6.282) (9.866) (10.133)(8.951) (8.810) Min, Max 154.9, 165.1, 152.5, 147.3, 157.0, 156.2,146.0, 155.0, 146.0, 146.0, 189.0 185.4 179.0 179.0 182.5 181.0 179.0190.5 190.5 190.5 Body mass index (kg/m²) Mean (SD) 27.04 26.35 26.8127.65 26.18 28.07 26.92 28.50 27.40 27.32 (3.924) (5.480) (3.278)(3.984) (5.173) (3.843) (3.712) (4.341) (4.093) (4.040) Min, Max 20.4,19.4, 21.1, 20.5, 18.5, 22.7, 20.6, 20.1, 18.5, 18.5, 34.7 32.5 32.634.6 34.5 34.8 35.0 34.7 35.0 35.0

Safety Solicited Events

Solicited adverse events were collected through 7 days after eachvaccination (Table 2 and Table 3). Across dose levels, the rate ofsolicited adverse events generally decreased between the first andsecond vaccination. Injection site pain was the most common solicitedlocal adverse event, with rates of 10-100% across treatment groups,which did not increase with dose level.

TABLE 2 Solicited Adverse Events by Grade and Treatment Group-FirstVaccination (First Vaccination Solicited Safety Set) hMPV/hPIV3 mRNAvaccine 25 μg 75 μg 150 μg 300 μg Placebo 2-Dose 1-Dose 2-Dose 1-Dose2-Dose 1-Dose 2-Dose Total (N = 30) (N = 4) (N = 13) (N = 17) (N = 13)(N = 17) (N = 13) (N = 17) (N = 94) n (%) n (%) n (%) n (%) n (%) n (%)n (%) n (%) n (%) Solicited Local AEs Pain-N1 28 4 13 17 13 17 13 17 94Any 3 3 12 14 10 15 13 13 80 (10.7) (75.0) (92.3) (82.4) (76.9) (88.2)(100.0) (76.5) (85.1) Grade 1 2 (7.1) 1 (25.0) 8 (61.5) 6 (35.3) 3(23.1) 7 (41.2) 2 (15.4) 6 (35.3) 33 (35.1) Grade 2 1 (3.6) 2 (50.0) 2(15.4) 7 (41.2) 5 (38.5) 7(41.2) 7 (53.8) 5 (29.4) 35 (37.2) Grade 3 0 02 (15.4) 1 (5.9) 2(15.4) 1 (5.9) 4 (30.8) 2 (11.8) 12(12.8) Erythema 274 11 15 11 15 12 16 84 (Redness)-N1 Any 0 0 0 2 (13.3) 0 0 0 1 (6.3) 3(3.6) Grade 1 0 0 0 1 (6.7) 0 0 0 1 (6.3) 2 (2.4) Grade 2 0 0 0 0 0 0 00 0 Grade 3 0 0 0 1 (6.7) 0 0 0 0 1 (1.2) Swelling 26 4 11 16 11 15 1216 85 (Hardness)-N1 Any 0 0 0 3 (18.8) 1 (9.1) 0 2 (16.7) 2 (12.5) 8(9.4) Grade 1 0 0 0 2 (12.5) 1 (9.1) 0 0 1 (6.3) 4 (4.7) Grade 2 0 0 0 00 0 1 (8.3) 1 (6.3) 2 (2.4) Grade 3 0 0 0 1 (6.3) 0 0 1 (8.3) 0 2 (2.4)Solicited Systemic AEs Fever-N1 30 4 13 17 13 17 13 17 94 Any 0 0 1(7.7) 1 (5.9) 4 (30.8) 0 4 (30.8) 4 (23.5) 14 (14.9) Grade 1 0 0 0 0 0 01 (7.7) 0 1 (1.1) Grade 2 0 0 0 1 (5.9) 3 (23.1) 0 3 (23.1) 4 (23.5) 11(11.7) Grade 3 0 0 1 (7.7) 0 1 (7.7) 0 0 0 2 (2.1) Headache-N1 27 4 1317 12 17 13 17 93 Any 3 (11.1) 1 (25.0) 6 (46.2) 5 (29.4) 7 (58.3) 8(47.1) 11 (84.6) 10 (58.8) 48 (51.6) Grade 1 3 (11.1) 1 (25.0) 5 (38.5)1 (5.9) 6 (50.0) 5 (29.4) 7 (53.8) 6 (35.3) 31 (33.3) Grade 2 0 0 0 4(23.5) 1 (8.3) 1 (5.9) 2 (15.4) 4 (23.5) 12 (12.9) Grade 3 0 0 1 (7.7) 00 2 (11.8) 2 (15.4) 0 5 (5.4) Fatigue-N1 27 4 13 17 11 17 13 17 92 Any 4(14.8) 1 (25.0) 3 (23.1) 5 (29.4) 8 (72.7) 6 (35.3) 10 (76.9) 11 (64.7)44 (47.8) Grade 1 4 (14.8) 1 (25.0) 1 (7.7) 2 (11.8) 6 (54.5) 1 (5.9) 3(23.1) 7(41.2) 21 (22.8) Grade 2 0 0 1 (7.7) 2 (11.8) 1 (9.1) 4 (23.5) 5(38.5) 4 (23.5) 17 (18.5) Grade 3 0 0 1 (7.7) 1 (5.9) 1 (9.1) 1 (5.9) 2(15.4) 0 6 (6.5) Myalgia-N1 27 4 13 17 11 17 13 17 92 Any 2 (7.4) 1(25.0) 4 (30.8) 2 (11.8) 7 (63.6) 5 (29.4) 9 (69.2) 11 (64.7) 39 (42.4)Grade 1 2 (7.4) 0 3 (23.1) 0 4 (36.4) 2 (11.8) 3 (23.1) 4 (23.5) 16(17.4) Grade 2 0 1 (25.0) 0 1 (5.9) 2 (18.2) 1 (5.9) 5 (38.5) 6 (35.3)16 (17.4) Grade 3 0 0 1 (7.7) 1 (5.9) 1 (9.1) 2 (11.8) 1 (7.7) 1 (5.9) 7(7.6) Arthralgia-N1 27 4 13 17 11 17 13 17 92 Any 2 (7.4) 1 (25.0) 2(15.4) 5 (29.4) 4 (36.4) 4 (23.5) 8 (61.5) 6 (35.3) 30 (32.6) Grade 1 2(7.4) 1 (25.0) 0 2 (11.8) 2 (18.2) 2 (11.8) 3 (23.1) 3 (17.6) 13 (14.1)Grade 2 0 0 2 (15.4) 2 (11.8) 2 (18.2) 1 (5.9) 3 (23.1) 3 (17.6) 13(14.1) Grade 3 0 0 0 1 (5.9) 0 1 (5.9) 2 (15.4) 0 4 (4.3) Nausea-N1 27 413 17 11 17 13 17 92 Any 0 0 3 (23.1) 3 (17.6) 4 (36.4) 3 (17.6) 4(30.8) 4 (23.5) 21 (22.8) Grade 1 0 0 2 (15.4) 1 (5.9) 2 (18.2) 1 (5.9)4 (30.8) 4 (23.5) 14 (15.2) Grade 2 0 0 0 2 (11.8) 2 (18.2) 1 (5.9) 0 05 (5.4) Grade 3 0 0 1 (7.7) 0 0 1 (5.9) 0 0 2 (2.2)

TABLE 3 Solicited Adverse Events by Grade and Treatment Group-SecondVaccination (Second Vaccination Solicited Safety Set) hMPV/hPIV3 mRNAvaccine 25 μg 75 μg 150 μg 300 μg Placebo 2-Dose 1-Dose 2-Dose 1-Dose2-Dose 1-Dose 2-Dose Total (N = 28) (N = 4) (N = 12) (N = 17) (N = 13)(N = 17) (N = 11) (N = 17) (N = 91) n (%) n (%) n (%) n (%) n (%) n (%)n (%) n (%) n (%) Solicited Local AEs Pain-N1 27 4 12 17 13 17 10 17 90Any 4 (14.8) 2 (50.0) 2 (16.7) 12 (70.6) 4 (30.8) 12 (70.6) 1 (10.0) 13(76.5) 46 (51.1) Grade 1 3 (11.1) 0 2 (16.7) 6 (35.3) 2 (15.4) 7 (41.2)1 (10.0) 7 (41.2) 25 (27.8) Grade 2 1 (3.7) 2 (50.0) 0 5 (29.4) 2 (15.4)4 (23.5) 0 5 (29.4) 18 (20.0) Grade 3 0 0 0 1 (5.9) 0 1 (5.9) 0 1 (5.9)3 (3.3) Erythema 26 4 12 15 13 17 9 17 87 (Redness)-N1 Any 1 (3.8) 0 0 1(6.7) 0 1 (5.9) 0 1 (5.9) 3 (3.4) Grade 1 0 0 0 1 (6.7) 0 0 0 1 (5.9) 2(2.3) Grade 2 0 0 0 0 0 1 (5.9) 0 0 1 (1.1) Grade 3 1 (3.8) 0 0 0 0 0 00 0 Swelling 26 4 12 15 13 17 9 17 87 (Hardness)-N1 Any 0 0 0 2 (13.3) 02 (11.8) 0 1 (5.9) 5 (5.7) Grade 1 0 0 0 2 (13.3) 0 2 (11.8) 0 1 (5.9) 5(5.7) Grade 2 0 0 0 0 0 0 0 0 0 Grade 3 0 0 0 0 0 0 0 0 0 SolicitedSystemic AEs Fever-N1 28 4 12 17 13 17 11 17 91 Any 0 0 0 0 0 1 (5.9) 02 (11.8) 3 (3.3) Grade 1 0 0 0 0 0 1 (5.9) 0 0 1 (1.1) Grade 2 0 0 0 0 00 0 1 (5.9) 1 (1.1) Grade 3 0 0 0 0 0 0 0 1 (5.9) 1 (1.1) Headache-N1 274 12 17 13 17 10 17 90 Any 2 (7.4) 1 (25.0) 1 (8.3) 5 (29.4) 4 (30.8) 9(52.9) 1 (10.0) 8 (47.1) 29 (32.2) Grade 1 1 (3.7) 1 (25.0) 1 (8.3) 3(17.6) 4 (30.8) 7 (41.2) 0 5 (29.4) 21 (23.3) Grade 2 1 (3.7) 0 0 2(11.8) 0 0 1 (10.0) 3 (17.6) 6 (6.7) Grade 3 0 0 0 0 0 2 (11.8) 0 0 2(2.2) Fatigue-N1 27 4 12 17 13 17 10 17 90 Any 2 (7.4) 0 0 4 (23.5) 4(30.8) 7 (41.2) 3 (30.0) 9 (52.9) 27 (30.0) Grade 1 1 (3.7) 0 0 2 (11.8)2 (15.4) 5 (29.4) 2 (20.0) 5 (29.4) 16 (17.8) Grade 2 1 (3.7) 0 0 1(5.9) 2 (15.4) 1 (5.9) 1 (10.0) 4 (23.5) 9 (10.0) Grade 3 0 0 0 1 (5.9)0 1 (5.9) 0 0 2 (2.2) Myalgia-N1 27 4 12 17 13 17 10 17 90 Any 2 (7.4) 1(25.0) 1 (8.3) 6 (35.3) 2 (15.4) 7 (41.2) 1 (10.0) 10 (58.8) 28 (31.1)Grade 1 2 (7.4) 0 1 (8.3) 4 (23.5) 1 (7.7) 4 (23.5) 1 (10.0) 5 (29.4) 16(17.8) Grade 2 0 1 (25.0) 0 1 (5.9) 1 (7.7) 2 (11.8) 0 5 (29.4) 10(11.1) Grade 3 0 0 0 1 (5.9) 0 1 (5.9) 0 0 2 (2.2) Arthralgia-N1 27 4 1217 13 17 10 17 90 Any 0 1 (25.0) 1 (8.3) 4 (23.5) 2 (15.4) 4 (23.5) 1(10.0) 8 (47.1) 21 (23.3) Grade 1 0 0 1 (8.3) 2 (11.8) 2 (15.4) 2 (11.8)1 (10.0) 5 (29.4) 13 (14.4) Grade 2 0 1 (25.0) 0 2 (11.8) 0 1 (5.9) 0 3(17.6) 7 (7.8) Grade 3 0 0 0 0 0 1 (5.9) 0 0 1 (1.1) Nausea-N1 27 4 1217 13 17 10 17 90 Any 1 (3.7) 0 0 4 (23.5) 0 4 (23.5) 1 (10.0) 5 (29.4)14 (15.6) Grade 1 1 (3.7) 0 0 4 (23.5) 0 3 (17.6) 1 (10.0) 4 (23.5) 12(13.3) Grade 2 0 0 0 0 0 1 (5.9) 0 1 (5.9) 2 (2.2) Grade 3 0 0 0 0 0 0 00 0

Unsolicited Events

Unsolicited events were collected through 28 days after each vaccination(Table 4 and Table 5). For subjects in the treatment groups, the mostcommon unsolicited AEs overall were upper respiratory tract infection,chills, and headache, which were reported by 5 (5.3%) subjects each.

No SAEs, AEs of special interest, or AEs leading to withdrawal werereported.

There was no pattern of clinically relevant lab abnormalities or changesfrom baseline lab values across vaccine treatment groups.

TABLE 4 Solicited Adverse Events by Grade and Treatment Group-FirstVaccination (First Vaccination Solicited Safety Set) hMPV/hPIV3 mRNAvaccine 25 μg 75 μg 150 μg 300 μg Placebo 2-Dose 1-Dose 2-Dose 1-Dose2-Dose 1-Dose 2-Dose Total (N = 30) (N = 4) (N = 13) (N = 17) (N = 13)(N = 17) (N = 13) (N = 17) (N = 94) n (%) n (%) n (%) n (%) n (%) n (%)n (%) n (%) n (%) Solicited Local AEs Pain-N1 28 4 13 17 13 17 13 17 94Any 3 (10.7) 3 (75.0) 12 (92.3) 14 (82.4) 10 (76.9) 15 (88.2) 13 (100.0)13 (76.5) 80 (85.1) Grade 1 2 (7.1) 1 (25.0) 8 (61.5) 6 (35.3) 3 (23.1)7 (41.2) 2 (15.4) 6 (35.3) 33(35.1) Grade 2 1 (3.6) 2 (50.0) 2 (15.4) 7(41.2) 5 (38.5) 7 (41.2) 7 (53.8) 5 (29.4) 35 (37.2) Grade 3 0 0 2(15.4) 1 (5.9) 2 (15.4) 1 (5.9) 4 (30.8) 2(11.8) 12(12.8) Erythema 27 411 15 11 15 12 16 84 (Redness)-N1 Any 0 0 0 2 (13.3) 0 0 0 1 (6.3) 3(3.6) Grade 1 0 0 0 1 (6.7) 0 0 0 1 (6.3) 2 (2.4) Grade 2 0 0 0 0 0 0 00 0 Grade 3 0 0 0 1 (6.7) 0 0 0 0 1 (1.2) Swelling 26 4 11 16 11 15 1216 85 (Hardness)-N1 Any 0 0 0 3 (18.8) 1 (9.1) 0 2 (16.7) 2 (12.5) 8(9.4) Grade 1 0 0 0 2 (12.5) 1 (9.1) 0 0 1 (6.3) 4 (4.7) Grade 2 0 0 0 00 0 1 (8.3) 1 (6.3) 2 (2.4) Grade 3 0 0 0 1 (6.3) 0 0 1 (8.3) 0 2 (2.4)Solicited Systemic AEs Fever-N1 30 4 13 17 13 17 13 17 94 Any 0 0 1(7.7) 1 (5.9) 4 (30.8) 0 4 (30.8) 4 (23.5) 14 (14.9) Grade 1 0 0 0 0 0 01 (7.7) 0 1 (1.1) Grade 2 0 0 0 1 (5.9) 3 (23.1) 0 3 (23.1) 4 (23.5) 11(11.7) Grade 3 0 0 1 (7.7) 0 1 (7.7) 0 0 0 2 (2.1) Headache-N1 27 4 1317 12 17 13 17 93 Any 3 (11.1) 1 (25.0) 6 (46.2) 5 (29.4) 7 (58.3) 8(47.1) 11 (84.6) 10 (58.8) 48 (51.6) Grade 1 3 (11.1) 1 (25.0) 5 (38.5)1 (5.9) 6 (50.0) 5 (29.4) 7 (53.8) 6 (35.3) 31 (33.3) Grade 2 0 0 0 4(23.5) 1 (8.3) 1 (5.9) 2 (15.4) 4 (23.5) 12 (12.9) Grade 3 0 0 1 (7.7) 00 2 (11.8) 2 (15.4) 0 5 (5.4) Fatigue-N1 27 4 13 17 11 17 13 17 92 Any 4(14.8) 1 (25.0) 3 (23.1) 5 (29.4) 8 (72.7) 6 (35.3) 10 (76.9) 11 (64.7)44 (47.8) Grade 1 4 (14.8) 1 (25.0) 1 (7.7) 2 (11.8) 6 (54.5) 1 (5.9) 3(23.1) 7 (41.2) 21 (22.8) Grade 2 0 0 1 (7.7) 2 (11.8) 1 (9.1) 4 (23.5)5 (38.5) 4 (23.5) 17 (18.5) Grade 3 0 0 1 (7.7) 1 (5.9) 1 (9.1) 1 (5.9)2 (15.4) 0 6 (6.5) Myalgia-N1 27 4 13 17 11 17 13 17 92 Any 2 (7.4) 1(25.0) 4 (30.8) 2 (11.8) 7 (63.6) 5 (29.4) 9 (69.2) 11 (64.7) 39 (42.4)Grade 1 2 (7.4) 0 3 (23.1) 0 4 (36.4) 2 (11.8) 3 (23.1) 4 (23.5) 16(17.4) Grade 2 0 1 (25.0) 0 1 (5.9) 2 (18.2) 1 (5.9) 5 (38.5) 6 (35.3)16 (17.4) Grade 3 0 0 1 (7.7) 1 (5.9) 1 (9.1) 2 (11.8) 1 (7.7) 1 (5.9) 7(7.6) Arthralgia-N1 27 4 13 17 11 17 13 17 92 Any 2 (7.4) 1 (25.0) 2(15.4) 5 (29.4) 4 (36.4) 4 (23.5) 8 (61.5) 6 (35.3) 30 (32.6) Grade 1 2(7.4) 1 (25.0) 0 2 (11.8) 2 (18.2) 2 (11.8) 3 (23.1) 3 (17.6) 13 (14.1)Grade 2 0 0 2 (15.4) 2 (11.8) 2 (18.2) 1 (5.9) 3 (23.1) 3 (17.6) 13(14.1) Grade 3 0 0 0 1 (5.9) 0 1 (5.9) 2 (15.4) 0 4 (4.3) Nausea-N1 27 413 17 11 17 13 17 92 Any 0 0 3 (23.1) 3 (17.6) 4 (36.4) 3 (17.6) 4(30.8) 4 (23.5) 21 (22.8) Grade 1 0 0 2 (15.4) 1 (5.9) 2 (18.2) 1 (5.9)4 (30.8) 4 (23.5) 14 (15.2) Grade 2 0 0 0 2 (11.8) 2 (18.2) 1 (5.9) 0 05 (5.4) Grade 3 0 0 1 (7.7) 0 0 1 (5.9) 0 0 2 (2.2)

TABLE 5 Solicited Adverse Events by Grade and Treatment Group-SecondVaccination (Second Vaccination Solicited Safety Set) hMPV/hPIV3 mRNAvaccine 25 μg 75 μg 150 μg 300 μg Placebo 2-Dose 1-Dose 2-Dose 1-Dose2-Dose 1-Dose 2-Dose Total (N = 28) (N = 4) (N = 12) (N = 17) (N = 13)(N = 17) (N = 11) (N = 17) (N = 94) n (%) n (%) n (%) n (%) n (%) n (%)n (%) n (%) n (%) Solicited Local AEs Pain-N1 27 4 12 17 13 17 10 17 90Any 4 (14.8) 2 (50.0) 2 (16.7) 12 (70.6) 4 (30.8) 12 (70.6) 1 (10.0) 13(76.5) 46 (51.1) Grade 1 3 (11.1) 0 2 (16.7) 6 (35.3) 2 (15.4) 7 (41.2)1 (10.0) 7 (41.2) 25 (27.8) Grade 2 1 (3.7) 2 (50.0) 0 5 (29.4) 2 (15.4)4 (23.5) 0 5 (29.4) 18 (20.0) Grade 3 0 0 0 1 (5.9) 0 1 (5.9) 0 1 (5.9)3 (3.3) Erythema 26 4 12 15 13 17 9 17 87 (Redness)-N1 Any 1 (3.8) 0 0 1(6.7) 0 1 (5.9) 0 1 (5.9) 3 (3.4) Grade 1 0 0 0 1 (6.7) 0 0 0 1 (5.9) 2(2.3) Grade 2 0 0 0 0 0 1 (5.9) 0 0 1 (1.1) Grade 3 1 (3.8) 0 0 0 0 0 00 0 Swelling 26 4 12 15 13 17 9 17 87 (Hardness)-N1 Any 0 0 0 2 (13.3) 02 (11.8) 0 1 (5.9) 5 (5.7) Grade 1 0 0 0 2 (13.3) 0 2 (11.8) 0 1 (5.9) 5(5.7) Grade 2 0 0 0 0 0 0 0 0 0 Grade 3 0 0 0 0 0 0 0 0 0 SolicitedSystemic AEs Fever-N1 28 4 12 17 13 17 11 17 91 Any 0 0 0 0 0 1 (5.9) 02 (11.8) 3 (3.3) Grade 1 0 0 0 0 0 1 (5.9) 0 0 1 (1.1) Grade 2 0 0 0 0 00 0 1 (5.9) 1 (1.1) Grade 3 0 0 0 0 0 0 0 1 (5.9) 1 (1.1) Headache-N1 274 12 17 13 17 10 17 90 Any 2 (7.4) 1 (25.0) 1 (8.3) 5 (29.4) 4 (30.8) 9(52.9) 1 (10.0) 8 (47.1) 29 (32.2) Grade 1 1 (3.7) 1 (25.0) 1 (8.3) 3(17.6) 4 (30.8) 7 (41.2) 0 5 (29.4) 21 (23.3) Grade 2 1 (3.7) 0 0 2(11.8) 0 0 1 (10.0) 3 (17.6) 6 (6.7) Grade 3 0 0 0 0 0 2 (11.8) 0 0 2(2.2) Fatigue-N1 27 4 12 17 13 17 10 17 90 Any 2 (7.4) 0 0 4 (23.5) 4(30.8) 7 (41.2) 3 (30.0) 9 (52.9) 27 (30.0) Grade 1 1 (3.7) 0 0 2 (11.8)2 (15.4) 5 (29.4) 2 (20.0) 5 (29.4) 16 (17.8) Grade 2 1 (3.7) 0 0 1(5.9) 2 (15.4) 1 (5.9) 1 (10.0) 4 (23.5) 9 (10.0) Grade 3 0 0 0 1 (5.9)0 1 (5.9) 0 0 2 (2.2) Myalgia-N1 27 4 12 17 13 17 10 17 90 Any 2 (7.4) 1(25.0) 1 (8.3) 6 (35.3) 2 (15.4) 7 (41.2) 1 (10.0) 10 (58.8) 28 (31.1)Grade 1 2 (7.4) 0 1 (8.3) 4 (23.5) 1 (7.7) 4 (23.5) 1 (10.0) 5 (29.4) 16(17.8) Grade 2 0 1 (25.0) 0 1 (5.9) 1 (7.7) 2 (11.8) 0 5 (29.4) 10(11.1) Grade 3 0 0 0 1 (5.9) 0 1 (5.9) 0 0 2 (2.2) Arthralgia-N1 27 4 1217 13 17 10 17 90 Any 0 1 (25.0) 1 (8.3) 4 (23.5) 2 (15.4) 4 (23.5) 1(10.0) 8 (47.1) 21 (23.3) Grade 1 0 0 1 (8.3) 2 (11.8) 2 (15.4) 2 (11.8)1 (10.0) 5 (29.4) 13 (14.4) Grade 2 0 1 (25.0) 0 2 (11.8) 0 1 (5.9) 0 3(17.6) 7 (7.8) Grade 3 0 0 0 0 0 1 (5.9) 0 0 1 (1.1) Nausea-N1 27 4 1217 13 17 10 17 90 Any 1 (3.7) 0 0 4 (23.5) 0 4 (23.5) 1 (10.0) 5 (29.4)14 (15.6) Grade 1 1 (3.7) 0 0 4 (23.5) 0 3 (17.6) 1 (10.0) 4 (23.5) 12(13.3) Grade 2 0 0 0 0 0 1 (5.9) 0 1 (5.9) 2 (2.2) Grade 3 0 0 0 0 0 0 00 0

Immunogenicity

All immunogenicity analyses were performed on the Per Protocol (PP)immunogenicity set, which included 118 of the 124 exposed subjects.

Baseline Neutralizing Antibody

Neutralizing antibodies against hMPV-A, hMPV-B, and PIV3 were present atbaseline (Day 1, prior to vaccination) in all subjects (Table 6). Thebaseline geometric mean titer (GMT) of neutralizing antibodies wasgenerally well balanced across treatment groups (Tables 7 and 8).

TABLE 6 Summary of Antibody Titers by Dose Group at Baseline (Day 1)(Per Protocol Immunogenicity Set) hMPV/hPIV3 mRNA vaccine Total Placebo25 μg 75 μg 150 μg 300 μg (mRNA) Total (N = 27) (N = 4) (N = 27) (N =29) (N = 29) (N = 89) (N = 116) hMPV-A Median 3694.0 3068.0 3310.02931.0 4407.0 3488.0 3582.5 Min, max 406, 860, 625, 455, 398, 398, 398,11806 6407 23216 11796 24084 24084 24084 GMT 2808.7 2464.9 2884.0 2974.83851.1 3178.6 3088.4 95% CI 1964.9, 537.9, 1969.4, 2250.0, 2579.4,2617.5, 2609.9, 4014.9 11294.8 4223.2 3933.2 5750.0 3860.0 3654.6 hMPV-BMedian 4214.0 4352.5 5522.0 2793.0 4865.0 4178.0 4194.5 Min, max 587,1786, 177, 688, 590, 177, 177, 23032 6042 562130 13138 236202 562130562130 GMT 3518.8 3779.7 6337.0 2926.8 6213.7 4783.0 4453.2 95% CI2277.4, 1641.1, 3025.3, 2133.5, 3570.2, 3533.6, 3462.7, 5436.8 8705.113274.0 4015.0 10814.8 6474.1 5727.0 PIV3 Median 380.0 328.5 345.0 352.0363.0 352.0 354.0 Min, max 92, 2692 179, 791 113, 3144 138, 2302 63,2678 63, 3144 63, 3144 GMT 374.2 336.3 341.6 359.0 450.5 379.7 378.4 95%CI 267.2, 108.3, 251.4, 278.7, 303.9, 318.7, 324.7, 523.9 1043.9 464.2462.5 667.6 452.3 441.0 N = number of subjects who meet per protocolanalysis at baseline (Day 1); GMT-geometric mean titer; CI-confidenceinterval

Neutralizing Antibody Response to First Vaccination

A single hMPV/hPIV3 mRNA vaccination boosted neutralizing antibodytiters against hMPV (lineages A and B) and PIV3 at all dose levelstested (25, 75, 150 and 300 μg) with no apparent dose response (FIG. 4). As shown in Table 7, the Day 28 (Month 1) to baseline GMR in the 75,150 and 300 μg dose groups ranged from 5.07-7.09 for hMPV-A, from4.87-7.73 for hMPV-B, and from 3.13-3.36 for PIV3, and the Day 28(Month 1) to baseline GMR for pooled hMPV/hPIV3 mRNA vaccination doselevels was 6.15 for hMPV-A, 6.36 for hMPV-B, and 3.29 for PIV3; and theseroresponse (percentage of subjects with >4× baseline titer) rangedfrom 51.9%-66.7% for hMPV-A, 59.3%-82.8% for hMPV-B, and 31.0%-51.7% forPIV3. A similar trend was observed in the 25 μg dose group of 4subjects. There was an inverse relationship between baselineneutralizing antibody titer and the response to the first hMPV/hPIV3mRNA vaccination (day 28/day 1 titer ratio), particularly for PIV3 (FIG.6 ). Thus, the hMPV/hPIV3 mRNA vaccination tended to induce a greaterboost in neutralizing antibody in subjects with lower baseline titers.There was no change in neutralizing antibody titer in the placebo groupfrom baseline to Day 28, translating to a GMR —1 and a 0% seroresponse,and suggesting absence of intercurrent hMPV or PIV3 infections duringthis time.

TABLE 7 Neutralizing Antibody by Dose Level and Visit Day; PPImmunogenicity Set Total Placebo 25 μg 75 μg 150 μg 300 μg (mRNA) N = 28N = 4 N = 27 N = 29 N = 29 N = 89 hMPV-A Day 1 GMT 2973.5 2464.9 2884.02974.8 3851.1 3178.6 Day 28 GMT 2967.1 15277.9 14617.9 17363.0 27306.819037.2 Day 28 GMR 1.00 8.52 5.07 5.84 7.09 6.04 Day 28 SR 0.0 66.7 51.962.1 65.5 60.2 hMPV-B Day 1 GMT 3640.1 3779.7 6337.0 2926.8 6213.74783.0 Day 28 GMT 3777.9 12904.8 30881.2 22626.0 43579.7 30307.8 Day 28GMR 1.04 3.53 4.87 7.73 7.01 6.33 Day 28 SR 0.0 66.7 59.3 82.8 62.1 68.2PIV3 Day 1 GMT 384.8 336.3 341.6 359.0 450.5 379.7 Day 28 GMT 396.61075.6 1149.3 1124.8 1482.0 1238.1 Day 28 GMR 1.03 2.67 3.36 3.13 3.293.24 Day 28 SR 0.00 33.3 37.0 31.0 51.7 39.8 N = number of subjects whomeet per protocol immunogenicity analysis definition at any timepoint;GMT-geometric mean titer; GMR-geometric mean ratio(post-baseline/baseline titer); SR-seroresponse = percentage of subjectswith >4 × baseline titer value at indicated time point.

Neutralizing Antibody Response to Second Vaccination

In the dose selection phase of the study, subjects in the 75 μg, 150 μgand 300 μg cohorts were randomly assigned in a 1:1 ratio to receive asecond dose of the hMPV/hPIV3 mRNA vaccine (2-dose groups) or placebo(1-dose groups) on Day 28. Within any given dose level, the 1-dose and2-dose cohorts might be expected to have similar GMT, GMR andseroresponse values at Day 28; however, this was not always the case.These differences are likely the result of smaller Ns when 1-dose and2-dose cohorts are analyzed separately (N=12-17, Table 8), than whencombined (N=28-29, Table 7).

For the hMPV-A and hMPV-B neutralizing antibody titers, the 95% CIs forthe GMR of the Month 2 titers to the Month 1 titers was approximately 1for the comparison at all dose levels. For the PIV3 neutralizingantibody titer, the ratio of the Month 2 titers to the Month 1 titerswas 1.51 for the 300 μg treatment group. This was the only treatmentgroup for PIV3 where the 95% CI for the GMR excluded 1. This suggeststhat the second vaccination did not impact the hMPV or PIV3 neutralizingantibody titers over this timeframe (FIG. 5 ).

TABLE 8 Neutralizing Antibody by Dose Level, Regimen (1-dose vs. 2-dose)and Visit Day; PP Immunogenicity Set 25 μg 75 μg 75 μg 150 μg 150 μg 300μg 300 μg Placebo 2-dose 1-dose 2-dose 1-dose 2-dose 1-dose 2-dose N =28 N = 4 N = 13 N = 14 N = 13 N = 16 N = 12 N = 17 hMPV-A GMT Day 12973.5 2464.9 3301.1 2543.9 3611.8 2541.0 3302.5 4292.4 Day 28 2967.115277.9 11715.6 17953.0 13964.7 20724.4 23915.5 29986.4 Day 56 3468.413483.0 12094.3 19240.8 11491.6 24962.5 19443.0 25165.1 Day 196 2738.99932.2 5052.2 7875.0 7265.7 11372.8 10687.1 14127.4 GMR Day 28 1.00 8.523.55 7.06 3.87 8.16 7.24 6.99 Day 56 1.17 7.52 3.87 7.56 3.18 9.82 6.055.86 Day 196 0.90 5.54 1.82 3.10 2.08 4.48 3.33 2.89 SR Day 28 0.00 66.738.5 64.3 53.8 68.8 66.7 64.7 Day 56 0.00 66.7 33.3 71.4 30.8 81.3 45.570.6 Day 196 0.00 66.7 27.3 35.7 25.0 62.5 45.5 37.5 hMPV-B GMT Day 13640.1 3779.7 3468.4 11090.2 3650.9 2445.6 6605.1 5951.5 Day 28 3777.912904.8 18980.2 48527.4 27912.8 19077.0 34427.8 51469.3 Day 56 3433.132382.7 12248.9 56084.6 24023.6 25900.1 29411.2 49424.8 Day 196 3905.58814.9 9894.8 23051.3 12785.3 15223.6 27230.9 28335.3 GMR Day 28 1.043.53 5.47 4.38 7.65 7.80 5.21 8.65 Day 56 0.94 8.86 4.14 5.06 6.58 10.594.24 8.30 Day 196 1.07 2.41 3.06 2.08 3.47 6.22 3.93 4.23 SR Day 28 0.0066.7 69.2 50.0 76.9 87.5 58.3 64.7 Day 56 0.00 66.7 41.7 50.0 76.9 93.845.5 76.5 Day 196 0.00 0.00 27.3 21.4 33.3 68.8 45.5 50.0 PIV3 GMT Day 1384.8 336.3 335.5 347.4 352.3 364.6 391.3 497.5 Day 28 396.6 1075.61385.5 966.2 1000.3 1237.3 1158.4 1763.5 Day 56 356.2 1387.8 1044.41250.5 692.9 1361.9 1121.6 2369.4 Day 196 280.2 599.1 541.3 651.2 431.8849.0 599.0 1786.5 GMR Day 28 1.03 2.67 4.13 2.78 2.84 3.39 2.96 3.54Day 56 0.93 3.45 3.34 3.60 1.97 3.74 3.23 4.76 Day 196 0.72 1.49 1.761.87 1.26 2.33 1.73 3.52 SR Day 28 0.00 33.3 46.2 28.6 23.1 37.5 50.052.9 Day 56 0.00 33.3 8.3 35.7 23.1 56.3 36.4 58.8 Day 196 0.00 0.00 9.121.4 0.00 18.8 18.2 43.8 N = number of subjects who meet per protocolimmunogenicity analysis definition at any timepoint; GMT-geometric meantiter; GMR-geometric mean ratio (post-baseline/baseline titer);SR-seroresponse = percentage of subjects with >4 × baseline titer valueat corresponding time point.

Neutralizing Antibody Persistence

Persistence of the neutralizing antibody response was evaluated at Month7 and Month 13. At Month 7, the neutralizing antibody GMT for allhMPV/hPIV3 mRNA vaccine dose levels was below the peak at Month 1 orMonth 2 but remained above baseline. Across the hMPV/hPIV3 mRNA vaccinedose levels, the Month 7 GMR ranged from 2.45 to 5.54 for hMPV-A, 2.41to 4.85 for hMPV B, and 1.49 to 2.63 for PIV3. The Month 7 GMR for thepooled hMPV/hPIV3 mRNA vaccine treatment groups was 2.98 for hMPV-A,3.65 for hMPV-B, and 2.03 for PIV3.

At Month 13, the hMPV neutralizing antibody GMT for all hMPV/hPIV3 mRNAvaccine dose levels remained above baseline. Across dose levels, the GMRranged from 1.50 to 3.88 for hMPV A and 1.18 to 3.82 for hMPV-B. TheMonth 13 GMR for the pooled hMPV/hPIV3 mRNA vaccine treatment groups was1.87 for hMPV-A and 2.91 for hMPV-B. At Month 13, the PIV3 neutralizingantibody GMT had generally returned to baseline. Across dose levels thePIV3 GMR ranged from 0.97 to 1.32 for PIV3, and was 1.06 for the pooledhMPV/hPIV3 mRNA vaccine treatment groups.

The dose level and regimen did not have a major impact on thepersistence of the neutralizing antibody response, although it is notedthat the Month 7 and Month 13 GMT was greatest in the 300 μg treatmentgroup for neutralizing antibodies against hMPV (both A and B lineages)and neutralizing antibodies against PIV3.

There was no increase in neutralizing antibody titer in the placebogroup from baseline to Month 7, reflected by a GMR —1 and a 0%seroresponse, and suggesting absence of intercurrent hMPV or PIV3infections during this time. This was also true for hMPV-A at Month 13.However, there was one subject in the placebo group for both hMPV-B andPIV3 with a seroresponse at Month 13.

Example 2. A Phase 1b, Randomized, Observer-Blind, Placebo-Controlled,Dose-Ranging Trial to Evaluate the Safety and Immunogenicity of aCombined Human Metapneumovirus (hMPV) and Parainfluenza Virus Type 3(PIV3) Vaccine when Administered to Adults, and to Children 12-36 Monthsof Age With Serologic Evidence of Prior Exposure Scientific Rationalefor Study Design

The design and dose levels proposed for this Phase 1b are based onobservations described in Example 1. Based on interim analysis to date,the hMPV/hPIV3 mRNA vaccine was generally well-tolerated in adults. Noserious adverse events (SAEs), adverse events (AEs) of special interest,or AEs leading to withdrawal were reported. There was no pattern ofclinically relevant laboratory abnormalities across treatment groups.Neutralizing antibodies against hMPV and PIV3 were present at baselinein all participants, consistent with prior exposure to both viruses.

The Phase 1b study evaluates 3 dose levels of the hMPV/hPIV3 mRNAvaccine for safety and immunogenicity in seropositive hMPV and PIV3children 12 to 36 months of age using a dose escalation design and isintended to support the progression to evaluation in seronegativechildren if tolerated in the seropositive children. See FIG. 8 . Alead-in cohort in healthy adults is also included to confirm the safetyprofile observed in Example 1.

The safety and tolerability of the hMPV/hPIV3 mRNA vaccine is firstevaluated in 15 participants 12-36 months of age at the lowest doselevel of 10 μg before sequential escalation to the planned higher doselevels of 30 and 100 μg. Enrollment of successive dose level cohortsfollows Safety Monitoring Committee (SMC) review and oversight in eachinstance.

Justification for the Choice of Study Population

The purpose of this study is to assess the safety and immunogenicity ofthe hMPV/hPIV3 mRNA vaccine in children 12-36 months of agecorresponding to a pediatric population closer in age to the primarytarget population where the disease burden still exists, howevergenerally considered to be less severe than in the very young infants.In an abundance of caution, participants are selected to be doublyseropositive to hMPV and PIV3 by microneutralization assay prior trialenrollment, to initiate the pediatric development in children who havehad previous infection of both hMPV and PIV3.

A lead-in cohort in healthy adults is also included to confirm thesafety profile observed in Example 1 in support of the implementation ofa minor manufacturing process change in the hMPV/hPIV3 mRNA vaccine.

Justification for the Dose and Schedule

In the Phase 1b study, the 2 dose levels of the hMPV/hPIV3 mRNA vaccinetested in adults are 30 μg (corresponding to one dose intended to betested in children), and 150 μg for comparison with one dose previouslytested in Example 1.

The dose range being tested in the toddlers in the Phase 1b study hasbeen selected based on the results of Example 1 and corresponds to amultiplying factor of approximately 3 starting with the lowest dose. Thelowest dose level in the Phase 1b trial at 10 μg is lower than thelowest level of 25 μg tested in Example 1, considering that youngchildren or infants reactogenicity may be more limiting.

Study Design

This is a Phase 1b, randomized, observer-blind, placebo-controlled,dose-ranging trial. The safety profile of the Adult Cohort permitsenrollment of the Pediatric Cohort. The Adult Cohort comprises healthyadults 18-49 years of age randomized in parallel 1:1:1 who receive oneof 2 dose levels of the hMPV/hPIV3 mRNA vaccine or placebo. ThePediatric Cohort comprises healthy children 12-36 months of agerandomized sequentially into 3 increasing dose levels of the hMPV/hPIV3mRNA vaccine, with each dose level randomized in a 1:1 ratio who receivethe hMPV/hPIV3 mRNA vaccine or placebo. A 2-vaccination, 0, 2-monthschedule is administered to all participants at all dose levels. Thetreatment dose levels are as follows:

Adult Cohort

Approximately 24 participants are randomized 1:1:1 to receive either 30μg of hMPV/hPIV3 mRNA vaccine, 150 μg of hMPV/hPIV3 mRNA vaccine, orplacebo.

Pediatric Cohort_(_)(enrolled sequentially following safety assessmentpost second vaccination before escalation to the next dose level):

Dose Level 1: 30 participants is randomized 1:1 to receive either 10 μgof hMPV/hPIV3 mRNA vaccine or placebo.

Dose Level 2: 30 participants is randomized 1:1 to receive either 30 μgof hMPV/hPIV3 mRNA vaccine or placebo.

Dose Level 3: 30 participants is randomized 1:1 to receive either 100 μgof hMPV/hPIV3 mRNA vaccine or placebo.

The primary purpose of this trial is to assess the safety andimmunogenicity of the hMPV/hPIV3 mRNA vaccine in adults and pediatricparticipants with serologic evidence of prior exposure to hMPV and PIV3.The dose levels of the hMPV/hPIV3 mRNA vaccine are based on the safetyand immunogenicity profile of the vaccine in the Phase 1 trial (Example1). Enrollment into the trial begins with an Adult Cohort followed byenrollment of a Pediatric Cohort.

Number of Participants

Approximately 24 adults and 90 children 12-36 months of age (114 total).

Inclusion Criteria

Adult and pediatric participants are eligible to be included in thestudy. Adults 18-49 years of age and children 12-36 months of age.

Exclusion Criteria

Adult and pediatric participants eligible for this study must not meetany of the following criteria: (1) Acutely ill or febrile (2) History ofa diagnosis or condition that may affect trial assessment or compromiseparticipant safety, specifically: Congenital or acquiredimmunodeficiency, including human immunodeficiency virus (HIV)infection. Chronic hepatitis, or suspected active hepatitis. A bleedingdisorder that is considered a contraindication to IM injection orphlebotomy. Dermatologic conditions that could affect local solicited ARassessments. Allergic or anaphylactic reactions following a vaccinationthat required medical intervention. Febrile seizures or recent receiptof inactivated vaccines or live virus vaccines or undergoing systemicimmunosuppression.

Investigational Product, Dosage, and Mode of Administration

The hMPV/hPIV3 mRNA vaccine injection consists of 2 distinct mRNAsequences that encode the full-length membrane-bound F proteins of hMPVand PIV3. The 2 mRNA Drug Substances are formulated at a target massratio of 1:1 in a mixture of 4 lipids to form a drug lipid complex LNP.The 4 lipids are heptadecan-9-yl8-((2-hydroxyethyl)(6-oxo-6(undecyloxy)hexyl)amino)octanoate (ionizablecationic lipid); 1,2-dimyristoyl-sn-glycerol, methoxypolyethyleneglycol(PEG2000-DMG); 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); andcholesterol.

The hMPV/hPIV3 mRNA vaccine injection is provided in 2-mL glass vials asa sterile liquid for injection and stored until use.

Estimated Study Duration

Adult participants are followed for up to approximately 8 months(approximately 6 months after the last vaccination). Pediatricparticipants are followed for up to approximately 13 months(approximately 11 months after the last vaccination).

Reference Therapy, Dosage, and Mode of Administration

Placebo consisting of a 0.9% sodium chloride (saline) injection isadministered intramuscularly.

Criteria for Evaluation Safety Assessments

In adult participants, solicited local adverse reactions (ARs) ofinjection site pain, erythema (redness), and swelling/induration(hardness), and solicited systemic ARs of fever, headache, fatigue,myalgia, arthralgia, nausea/vomiting, chills and rash are assessed.Solicited local and systemic ARs occurring during the 7 days followingeach vaccination (the day of vaccination and 6 subsequent days) arerecorded by the participant via an electronic Diary (eDiary).

In pediatric participants, solicited local ARs of tenderness, erythema(redness) and swelling/induration (hardness), and solicited systemic ARsof fever, sleepiness, loss of appetite, chills/shivering,irritability/fussiness/persistent crying and rash are assessed.Solicited local and systemic ARs occurring during the 7 days followingeach vaccination (i.e., the day of vaccination and 6 subsequent days)are recorded.

Immunogenicity Assessments

GMT of serum anti-hMPV and anti-PIV3 neutralizing antibodies and GMR ofpost-baseline/baseline titers.

Proportion of participants with ≥2-fold and ≥4-fold increases in serumanti-hMPV or anti-PIV3 neutralizing antibody titer from baseline.

Exploratory assays to characterize the immune response to hMPV, PIV3 orother respiratory viruses are performed with excess serum.

SEQUENCES

It should be understood that any of the mRNA sequences described hereinmay include a 5′ UTR and/or a 3′ UTR. The UTR sequences may be selectedfrom the following sequences, or other known UTR sequences may be used.It should also be understood that any of the mRNA constructs describedherein may further comprise a polyA tail and/or cap (e.g.,7mG(5′)ppp(5′)NlmpNp). Further, while many of the mRNAs and encodedantigen sequences described herein include a signal peptide and/or apeptide tag (e.g., C-terminal His tag), it should be understood that theindicated signal peptide and/or peptide tag may be substituted for adifferent signal peptide and/or peptide tag, or the signal peptideand/or peptide tag may be omitted.

5′ UTR: (SEQ ID NO: 3) GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC5′ UTR: (SEQ ID NO: 4)GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGACCCCGGCGC CGCCACC 3′ UTR:(SEQ ID NO: 5) UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAA UAAAGUCUGAGUGGGCGGC3′ UTR: (SEQ ID NO: 6)UGAUAAUAGGCUGGAGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAA UAAAGUCUGAGUGGGCGGC

SEQ ID hMPV F Glycoprotein NO:SEQ ID NO: 1 consists of from 5′ end to 3′ end, 5′ UTR SEQ ID NO: 3, mRNA ORF SEQ ID1 NO: 7 , and 3′ UTR SEQ ID NO: 5. Chemistry 1-methylpseudouridine Cap7mG(5′)ppp(5′)NlmpNp 5′ UTR GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUA 3AGAGCCACC ORF of mRNA AUGAGCUGGAAGGUGGUGAUUAUCUUCAGCCUGCUGAUU 7Construct ACACCUCAACACGGCCUGAAGGAGAGCUACCUGGAAGAG (excluding the stopAGCUGCUCCACCAUCACCGAGGGCUACCUGAGCGUGCUGC codon)GGACCGGCUGGUACACCAACGUGUUCACCCUGGAGGUGGGCGACGUGGAGAACCUGACCUGCAGCGACGGCCCUAGCCUGAUCAAGACCGAGCUGGACCUGACCAAGAGCGCUCUGAGAGAGCUGAAGACCGUGUCCGCCGACCAGCUGGCCAGAGAGGAACAGAUCGAGAACCCUCGGCAGAGCAGAUUCGUGCUGGGCGCCAUCGCUCUGGGAGUCGCCGCUGCCGCUGCAGUGACAGCUGGAGUGGCCAUUGCUAAGACCAUCAGACUGGAAAGCGAGGUGACAGCCAUCAACAAUGCCCUGAAGAAGACCAACGAGGCCGUGAGCACCCUGGGCAAUGGAGUGAGAGUGCUGGCCACAGCCGUGCGGGAGCUGAAGGACUUCGUGAGCAAGAACCUGACCAGAGCCAUCAACAAGAACAAGUGCGACAUCGAUGACCUGAAGAUGGCCGUGAGCUUCUCCCAGUUCAACAGACGGUUCCUGAACGUGGUGAGACAGUUCUCCGACAACGCUGGAAUCACACCUGCCAUUAGCCUGGACCUGAUGACCGACGCCGAGCUGGCUAGAGCCGUGCCCAACAUGCCCACCAGCGCUGGCCAGAUCAAGCUGAUGCUGGAGAACAGAGCCAUGGUGCGGAGAAAGGGCUUCGGCAUCCUGAUUGGGGUGUAUGGAAGCUCCGUGAUCUACAUGGUGCAGCUGCCCAUCUUCGGCGUGAUCGACACACCCUGCUGGAUCGUGAAGGCCGCUCCUAGCUGCUCCGAGAAGAAAGGAAACUAUGCCUGUCUGCUGAGAGAGGACCAGGGCUGGUACUGCCAGAACGCCGGAAGCACAGUGUACUAUCCCAACGAGAAGGACUGCGAGACCAGAGGCGACCACGUGUUCUGCGACACCGCUGCCGGAAUCAACGUGGCCGAGCAGAGCAAGGAGUGCAACAUCAACAUCAGCACAACCAACUACCCCUGCAAGGUGAGCACCGGACGGCACCCCAUCAGCAUGGUGGCUCUGAGCCCUCUGGGCGCUCUGGUGGCCUGCUAUAAGGGCGUGUCCUGUAGCAUCGGCAGCAAUCGGGUGGGCAUCAUCAAGCAGCUGAACAAGGGAUGCUCCUACAUCACCAACCAGGACGCCGACACCGUGACCAUCGACAACACCGUGUACCAGCUGAGCAAGGUGGAGGGCGAGCAGCACGUGAUCAAGGGCAGACCCGUGAGCUCCAGCUUCGACCCCAUCAAGUUCCCUGAGGACCAGUUCAACGUGGCCCUGGACCAGGUGUUUGAGAACAUCGAGAACAGCCAGGCCCUGGUGGACCAGAGCAACAGAAUCCUGUCCAGCGCUGAGAAGGGCAACACCGGCUUCAUCAUUGUGAUCAUUCUGAUCGCCGUGCUGGGCAGCUCCAUGAUCCUGGUGAGCAUCUUCAUCAUUAUCAAGAAGACCAAGAAACCCACCGGAGCCCCUCCUGAGCUGAGCGGCGUGACCAACAAUGGCUUC AUUCCCCACAACUGA 3′ UTRUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCC 5CCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGG C Corresponding aminoMSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWY 8 acid sequenceTNVFTLEVGDVENLTCSDGPSLIKTELDLTKSALRELKTVSADQLAREEQIENPRQSRFVLGAIALGVAAAAAVTAGVAIAKTIRLESEVTAINNALKKTNEAVSTLGNGVRVLATAVRELKDFVSKNLTRAINKNKCDIDDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVPNMPTSAGQIKLMLENRAMetVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPEDQFNVALDQVFENIENSQALVDQSNRILSSAEKGNTGFIIVIILIAVLGSSMILVSIFIIIKKT KKPTGAPPELSGVTNNGFIPHNPolyA tail 100 nt SEQ ID hPIV3 F Glycoprotein NO:SEQ ID NO: 2 consists of from 5′ end to 3′ end, 5′ UTR SEQ ID NO: 3, mRNA ORF SEQ ID2 NO: 9 , and 3′ UTR SEQ ID NO: 5. Chemistry 1-methylpseudouridine Cap7mG(5′)ppp(5′)NlmpNp 5′ UTR GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUA 3AGAGCCACC ORF of mRNA AUGCCCAUCAGCAUCCUGCUGAUCAUCACCACAAUGAUC 9Construct AUGGCCAGCCACUGCCAGAUCGACAUCACCAAGCUGCAGC (excluding the stopACGUGGGCGUGCUCGUGAACAGCCCCAAGGGCAUGAAGA codon)UCAGCCAGAACUUCGAGACACGCUACCUGAUCCUGAGCCUGAUCCCCAAGAUCGAGGACAGCAACAGCUGCGGCGACCAGCAGAUCAAGCAGUACAAGCGGCUGCUGGACAGACUGAUCAUCCCCCUGUACGACGGCCUGCGGCUGCAGAAAGACGUGAUCGUGACCAACCAGGAAAGCAACGAGAACACCGACCCCCGGACCGAGAGAUUCUUCGGCGGCGUGAUCGGCACAAUCGCCCUGGGAGUGGCCACAAGCGCCCAGAUUACAGCCGCUGUGGCCCUGGUGGAAGCCAAGCAGGCCAGAAGCGACAUCGAGAAGCUGAAAGAGGCCAUCCGGGACACCAACAAGGCCGUGCAGAGCGUGCAGUCCAGCGUGGGCAAUCUGAUCGUGGCCAUCAAGUCCGUGCAGGACUACGUGAACAAAGAAAUCGUGCCCUCUAUCGCCCGGCUGGGCUGUGAAGCUGCCGGACUGCAGCUGGGCAUUGCCCUGACACAGCACUACAGCGAGCUGACCAACAUCUUCGGCGACAACAUCGGCAGCCUGCAGGAAAAGGGCAUUAAGCUGCAGGGAAUCGCCAGCCUGUACCGCACCAACAUCACCGAGAUCUUCACCACCAGCACCGUGGAUAAGUACGACAUCUACGACCUGCUGUUCACCGAGAGCAUCAAAGUGCGCGUGAUCGACGUGGACCUGAACGACUACAGCAUCACCCUGCAAGUGCGGCUGCCCCUGCUGACCAGACUGCUGAACACCCAGAUCUACAAGGUGGACAGCAUCUCCUACAACAUCCAGAACCGCGAGUGGUACAUCCCUCUGCCCAGCCACAUUAUGACCAAGGGCGCCUUUCUGGGCGGAGCCGACGUGAAAGAGUGCAUCGAGGCCUUCAGCAGCUACAUCUGCCCCAGCGACCCUGGCUUCGUGCUGAACCACGAGAUGGAAAGCUGCCUGAGCGGCAACAUCAGCCAGUGCCCCAGAACCACCGUGACCUCCGACAUCGUGCCCAGAUACGCCUUCGUGAAUGGCGGCGUGGUGGCCAACUGCAUCACCACCACCUGUACCUGCAACGGCAUCGGCAACCGGAUCAACCAGCCUCCCGAUCAGGGCGUGAAGAUUAUCACCCACAAAGAGUGUAACACCAUCGGCAUCAACGGCAUGCUGUUCAAUACCAACAAAGAGGGCACCCUGGCCUUCUACACCCCCGACGAUAUCACCCUGAACAACUCCGUGGCUCUGGACCCCAUCGACAUCUCCAUCGAGCUGAACAAGGCCAAGAGCGACCUGGAAGAGUCCAAAGAGUGGAUCCGGCGGAGCAACCAGAAGCUGGACUCUAUCGGCAGCUGGCACCAGAGCAGCACCACCAUCAUCGUGAUCCUGAUUAUGAUGAUUAUCCUGUUCAUCAUCAACAUUACCAUCAUCACUAUCGCCAUUAAGUACUACCGGAUCCAGAAACGGAACCGGGUGGACCAGAAUGACAAGCCCUACGUGCUGAC AAACAAG 3′ UTRUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCC 5CCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGG C Corresponding aminoMPISILLIITTMIMASHCQIDITKLQHVGVLVNSPKGMKISQNFE 10 acid sequenceTRYLILSLIPKIEDSNSCGDQQIKQYKRLLDRLIIPLYDGLRLQKDVIVTNQESNENTDPRTERFFGGVIGTIALGVATSAQITAAVALVEAKQARSDIEKLKEAIRDTNKAVQSVQSSVGNLIVAIKSVQDYVNKEIVPSIARLGCEAAGLQLGIALTQHYSELTNIFGDNIGSLQEKGIKLQGIASLYRTNITEIFTTSTVDKYDIYDLLFTESIKVRVIDVDLNDYSITLQVRLPLLTRLLNTQIYKVDSISYNIQNREWYIPLPSHIMTKGAFLGGADVKECIEAFSSYICPSDPGFVLNHEMESCLSGNISQCPRTTVTSDIVPRYAFVNGGVVANCITTTCTCNGIGNRINQPPDQGVKIITHKECNTIGINGMLFNTNKEGTLAFYTPDDITLNNSVALDPIDISIELNKAKSDLEESKEWIRRSNQKLDSIGSWHQSSTTIIVILIMMIILFIINITIITIAIKYYRIQKRNRVDQNDKPYV LTNK PolyA tail 100 nt

EQUIVALENTS

All references, patents and patent applications disclosed herein areincorporated by reference with respect to the subject matter for whicheach is cited, which in some cases may encompass the entirety of thedocument.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.” It should also beunderstood that, unless clearly indicated to the contrary, in anymethods claimed herein that include more than one step or act, the orderof the steps or acts of the method is not necessarily limited to theorder in which the steps or acts of the method are recited.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” “composed of,” and the like are tobe understood to be open-ended, i.e., to mean including but not limitedto. Only the transitional phrases “consisting of” and “consistingessentially of” shall be closed or semi-closed transitional phrases,respectively, as set forth in the United States Patent Office Manual ofPatent Examining Procedures, Section 2111.03.

The terms “about” and “substantially” preceding a numerical valuemean±10% of the recited numerical value.

Where a range of values is provided, each value between the upper andlower ends of the range are specifically contemplated and describedherein.

The entire contents of International Application Nos. PCT/US2015/02740,PCT/US2016/043348, PCT/US2016/043332, PCT/US2016/058327,PCT/US2016/058324, PCT/US2016/058314, PCT/US2016/058310,PCT/US2016/058321, PCT/US2016/058297, PCT/US2016/058319, andPCT/US2016/058314 are incorporated herein by reference.

1-58. (canceled)
 59. A method of inducing in a human subject humanmetapneumovirus (hMPV) neutralizing antibody titers, the methodcomprising administering to the human subject a vaccine compositioncomprising (a) a messenger RNA (mRNA) comprising an open reading frameencoding a wild-type hMPV fusion (F) glycoprotein, wherein the mRNAcomprises a nucleic acid sequence having at least 80% identity to thenucleic acid sequence of SEQ ID NO: 1, and (b) a lipid nanoparticle. 60.The method of claim 59, wherein the vaccine composition comprises a 25μg to 150 μg dose of the mRNA.
 61. The method of claim 60, wherein thevaccine composition comprises a 150 μg dose of the mRNA.
 62. The methodof claim 60, wherein the vaccine composition comprises a 75 μg dose ofthe mRNA.
 63. The method of claim 60, wherein the vaccine compositioncomprises a 25 μg dose of the mRNA.
 64. The method of claim 59, whereinthe lipid nanoparticle comprises an ionizable cationic lipid, anon-cationic lipid, a sterol, and a polyethylene glycol (PEG)-modifiedlipid.
 65. The method of claim 64, wherein the lipid nanoparticlecomprises 45-55 mole percent (mol %) of the ionizable cationic lipid,5-15 mol % of the non-cationic lipid, 35-45 mol % of the sterol, and 1-2mol % of the PEG-modified lipid.
 66. The method of claim 65, wherein theionizable cationic lipid is Compound I, the non-cationic lipid is DSPC(1,2-Distearoyl-sn-glycero-3-phosphocholine), the sterol is cholesterol,and the PEG-modified lipid is DMG-PEG(1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000), andwherein Compound I has the formula:


67. The method of claim 59, wherein the mRNA has at least 90% identityto the nucleic acid sequence of SEQ ID NO:
 1. 68. The method of claim67, wherein the mRNA has at least 95% identity to the nucleic acidsequence of SEQ ID NO:
 1. 69. The method of claim 68, wherein the mRNAcomprises the nucleic acid sequence of SEQ ID NO:
 1. 70. The method ofclaim 59, wherein the open reading frame sequence comprises the nucleicacid sequence of SEQ ID NO:
 7. 71. The method of claim 59, wherein thewild-type hMPV fusion (F) glycoprotein comprises the amino acid sequenceof SEQ ID NO:
 8. 72. The method of claim 59, wherein the mRNA comprises1-methylpseudourine.
 73. The method of claim 72, wherein the mRNAcomprises 1-methylpseudourine at all uridine positions of the mRNA. 74.The method of claim 59, wherein the mRNA comprises a 5′ cap and a polyAtail.
 75. The method of claim 59, wherein the mRNA comprises a 5′untranslated region and a 3′ untranslated region.
 76. A method ofinducing in a human subject human metapneumovirus (hMPV) neutralizingantibody titers, the method comprising administering to the humansubject a 25 μg to 150 μg dose of a vaccine composition comprising (a) amessenger RNA (mRNA) comprising an open reading frame encoding awild-type hMPV fusion (F) glycoprotein, wherein the mRNA comprises anucleic acid sequence having at least 80% identity to the nucleic acidsequence of SEQ ID NO: 1, and (b) a lipid nanoparticle, and wherein themRNA comprises 1-methylpseudourine.
 77. The method of claim 76, whereinthe mRNA comprises 1-methylpseudourine at all uridine positions of themRNA.
 78. The method of claim 77, wherein the mRNA further comprise a 5′untranslated region, 5′ cap, a polyA tail, and 3′ untranslated region.